Methods for treating neurological deficits

ABSTRACT

The present invention features methods and compositions for treating a patient who has a neurological deficit. The method can be carried out, for example, by contacting (in vivo or in culture) a neural progenitor cell of the patient&#39;s central nervous system (CNS) with a polypeptide that binds the epidermal growth factor (EGF) receptor and directing progeny of the proliferating progenitor cells to migrate en masse to a region of the CNS in which they will reside and function in a manner sufficient to reduce the neurological deficit. The method may include a further step in which the progeny of the neural precursor cells are contacted with a compound that stimulates differentiation.

[0001] This application claims benefit from provisional application Ser.No. 60/055,383, filed Aug. 4, 1997, which is hereby incorporated byreference in its entirety.

[0002] The field of the invention is treatment of neurological deficitscaused by an injury, disease, or developmental disorder that affects thecentral nervous system.

BACKGROUND OF THE INVENTION

[0003] Neurotrophic factors are peptides that variously support thesurvival, proliferation, differentiation, size, and function of nervecells (for review, see Loughlin and Fallon, Neurotrophic Factors,Academic Press, San Diego, Calif., 1993). While the numbers ofidentified trophic factors, or growth factors, are ever-increasing, mostcan be assigned to one or another established family based upon theirstructure or binding affinities. Growth factors from various families,including the epidermal growth factor (EGF) family, have beendemonstrated to support dopaminergic neurons of the nigrostriatal system(an area that can be treated according to the methods of the presentinvention) (for review, see Hefti, J. Neurobiol. 25:1418-1435, 1994;Unsicker, Prog. Growth Factor Res. 5:73-87, 1994).

[0004] EGF, the founding member of the EGF family, was characterizedmore than 25 years, ago (Savage and Cohen, J. Biol. Chem. 247:7609-7611,1972; Savage et al., J. Biol. Chem. 247:7612-7621, 1972). Since then,additional members have been identified; they include vaccinia virusgrowth factor.(VGF; Ventatesan et al., J. Virol. 44:637-646, 1982),myxomavirus growth factor (MGF; Upton et al., J. Virol. 61:1271-1275,1987), Shope fibroma virus growth factor (SFGF; Chang et al., Mol. Cell.Biol. 7:535-540, 1987), amphiregulin (A R; Kimura et al., Nature348:257-260, 1990), and heparin-binding EGF-like growth factor (HB-EGF;Higashiyama et al., Science 251:936-939, 1991). A common feature ofthese factors is an amino acid sequence containing six cysteines thatform three disulfide cross links and support a conserved structure thatunderlies their common ability to bind the EGF receptor.

[0005] EGF is by far the most-studied member of the family and was thefirst localized to brain tissue: EGF-like immunoreactivity (IR) wasfound in areas of developing adult forebrain and midbrain including theglobus pallidus, ventral pallidum, entopeduncular nucleus, substantianigra, and the Islands of Calleja (Fallon et al., Science 224:1107-1109,1984).

[0006] Another member of the EGF family, TGFα, has also been localizedto brain tissue. It binds the EGF receptor (Todaro et al., Proc. Natl.Acad. Sci. USA 77:5258-5262, 1980), stimulates the receptor's tyrosinekinase activity, and elicits similar mitogenic responses in a widevariety of cell types (for review, see Derynck, Adv. Cancer Res.58:27-52, 1992). TGFα might also bind to additional, unidentifiedreceptors (which may help explain its differential actions in somecells). TGFα-IR has previously been shown to be heterogeneouslydistributed in neuronal perikarya throughout the adult rat CNS and in asubpopulation of forebrain astrocytes (Code et al., Brain Res.421:401-405, 1987; Fallon et al., Growth Factors 2:241-250, 1990). TGFαmRNA has been detected in whole rodent brain (Lee et al., Mol. Cell.Biol. 5:3655-3646, 1985; Kudlow et al., J. Biol. Chem. 264:3880-3883,1989) and in striatum and other brain regions by a nuclease protectionassay (Weickert and Blum, Devel. Brain Res. 86:203-216, 1995) and by insitu nucleic acid hybridization (Seroogy et al., Neuroreport 6:105-108,1994).

[0007] TGFα and EGF mRNAs reach their highest relative abundance(compared to total RNA) in the early postnatal period and decreasethereafter, suggesting a role in development (Lee et al., 1985, supra;Lazar and Blum, J. Neurosci. 12:1688-1697, 1992). In whole brain, thereduction is over 50% (Lazar and Blum, 1992, supra), whereas, instriatum, relative TGFα mRNA drops by two-thirds from peak levels(Weickert and Blum, 1995, supra). At all developmental stages examined,whole brain TGFα mRNA exceeds EGF mRNA levels by more than an order ofmagnitude (Lazar and Blum, 1992, supra).

[0008] The EGF receptor was localized by immunocytochemistry toastrocytes and subpopulations of cortical and cerebellar neurons in ratbrain and to neurons in human autopsy brain specimens (Gomez-Pinilla etal., Brain Res. 438:385-390, 1988; Werner et al., J. Histochem.Cytochem. 36:81-86, 1988). EGF binding sites were revealed in ratcortical and subcortical areas, including the striatum, in anautoradiography study with radiolabeled EGF (Quirion et al., Synapse2:212-218, 1988). In situ hybridization studies demonstrated EGFreceptor mRNA in striatum and cells of the ventral mesencephalon(Seroogy et al., 1994, supra) and in proliferative regions in developingand adult rat brain (Seroogy et al., Brain Res. 670:157-164, 1995). Aswith relative EGF and TGFα mRNAs, EGF receptor mRNA is most abundant instriatum and ventral midbrain early in development, and graduallydeclines as the animal matures (Seroogy et al., 1994, supra).

[0009] Physiologically, TGFα acts on numerous cell types throughout thebody, including many of neural origin (for review, see Derynck, 1992,supra). It supports the survival of cultured central neurons (Morrisonet al., Science 238:72-75, 1987; Zhang et al., Cell. Regul. 1:511-521,1990) and, unlike EGF, enhances survival and neurite outgrowth of dorsalroot ganglion sensory neurons (Chalazonitis et al., J. Neurosci.12:583-594, 1992). It also stimulates proliferation and differentiationof neuronal and glial progenitor cells from developing and adult brains(Anchan et al., Neuron 6:923-936, 1991).

[0010] The trophic effects of EGF-family peptides on mesencephalicdopaminergic neurons in culture have also been studied in recent years.EGF enhances the survival of E16 dopamine neurons in mixed midbraincultures (Casper et al., J. Neurosci. Res. 30:372-381, 1991), but thedegree to which it stimulates dopamine uptake is modest (Knusel et al.,J. Neurosci. 10:558-570, 1990). TGFα also supports the survival ofmesencephalic dopamine neurons in dissociated cell culture, but itseffect is more selective than that of EGF (Ferrari et al., J. Neurosci.Res. 30:493-497, 1991; Alexi and Hefti, Neurosci. 55:903-918, 1993).

[0011] Another important characteristic of EGF-family growth factors istheir ability to protect midbrain dopamine cells from neurotoxicassaults. EGF has been shown to protect dopamine neurons from glutamateor quisqualate excitotoxicity in dissociated cell culture (Casper andBlum, J. Neurochem. 65:1016-1026, 1995). It has also been demonstratedto protect cultured dopamine cells from the selective dopamineneurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺; Park et al., Brain Res.599:83-97, 1992) and to increase dopamine uptake in MPP⁺-treatedcultures (Hadjiconstantinou et al., J. Neurochem. 57:479-482, 1991).

[0012] The results of studies with EGF in vivo were consistent thoseobtained in culture; EGF effected neuroprotection in both instances. Forexample, intracerebroventricular (ICV) infusions of EGF reducedamphetamine-induced rotations, increased the number of survivingtyrosine hydroxylase immunoreactive (TH-IR) cells in the SN, andincreased striatal TH-IR fibers after transection of the nigrostriatalpathway in a rat model of PD (Pezzoli et al., Movement Disord.6:281-287, 1993; Ventrella, J. Neurosurg. Sci. 37:1-8, 1993). ICVinfusions of EGF into the brains of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned miceenhanced the content of dopamine and 3,4-dihydro-zyphenylacetic acid(DOPAC) and the activity of tyrosine hydroxylase in the striatum(Hadjiconstantinou et al., 1991, supra; Schneider et al., Brain Res.674:260-264, 1995).

[0013] Despite its more potent activity in vitro, relative to EGF, thetrophic effects of TGFα in vivo—particularly in animals, includinghumans, with neurological deficits—are undetermined. The presentinvention is based on newly discovered effects of TGFα infusion on cellsin the normal and abnormal (lesioned) central nervous system, which aredescribed herein.

SUMMARY OF THE INVENTION

[0014] The present invention features methods and compositions fortreating a patient who has a neurological deficit. The method can becarried out, for example, by contacting (in vivo or in culture) a neuralprogenitor cell of the patient's central nervous system (CNS) with apolypeptide that binds the epidermal growth factor (EGF) receptor anddirecting progeny of the proliferating progenitor cells to migrate enmasse to a region of the CNS in which they will reside and function in amanner sufficient to reduce the neurological deficit. The method mayinclude a further step in which the progeny of the neural precursorcells are contacted with a compound that stimulates differentiation.

[0015] Other objects, advantages, and novel features of the presentinvention will become apparent from the brief description of thedrawings, the detailed description of the invention, and the workingexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic of a coronal section of rat forebrain. Theinjection pipette illustrated on the right-hand side represents onelocation into which a growth factor can be infused, the striatum (str).(cerebral cortex=ctx; lateral ventricle=lv).

[0017] FIGS. 2A-2D are photomicrographs of coronal sections through themesencephalon of the adult rat brain that have been probed to show thedistribution of EGF receptor mRNA. In FIG. 2A a “sense” probe wasapplied as a control. In FIGS. 2B-2D, sections through the substantianigra (sn) reveal moderately abundant expression in the hippocampus(hip), the medial portion of the sna, and the parabranchial andparanigral nuclei of the ventral tegmental area (vta). In themost-caudal midbrain (FIG. 2D), the interpeduncular nucleus (ip) was themost intensely labeled. Scale bar=5 mm.

[0018]FIG. 3 is a photomicrograph of a coronal section through thestriatum of an adult rat brain that was infused with TGFα and probed toshow the distribution of EGF receptor mRNA. On the side of the infusion,a dramatic increase in hybridization density is apparent in the medialstriatum adjacent to the lateral ventricle.

[0019]FIG. 4 is a photomicrograph of a coronal section through theforebrain of an adult rat brain that was infused with TGFα and lesionedwith 6-OHDA. In situ hybridization was performed to localize EGFreceptor mRNA, which appears as an intense ridge extending well into thebody of the striatum.

[0020]FIG. 5 is a bar graph showing the average standardized densitiesof TGFα mRNA hybridization in striata in each of five groups of animalsexamined (normal; aCSF infusion, no lesion; aCSF infusion, lesion; TGFαinfusion, no lesion; TGFα infusion, lesion). The paired bars representdensities in striata ipsilateral and contralateral to the treatment.Average hybridization density was significantly reduced by one-quarteripsilateral to the treatments in both groups receiving nigral 6-OHDAlesions. The striatal infusion of TGFα peptide had no impact on thedecrease. Averages±S.E.M. (Student's t-test, paired foripsilateral-contralateral comparisons; P values, * p<0.005, ** p<0.001).

[0021]FIG. 6 is a bar graph showing the average standardized densitiesof EGF receptor mRNA hybridization in the subependymal regions along theedges of the striata bordering the lateral ventricles of animals in thesame five test groups described in FIG. 5. The paired bars representdensities in striata ipsilateral and contralateral to the treatment.Average hybridization density was approximately doubled in theipsilateral subependymal region in both groups receiving TGFα striatalinfusions. Averages±S.E.M. (Student's t-test, paired foripsilateral-contralateral comparisons; P values, * p<0.01, ** p<0.0001).

[0022]FIG. 7 is a bar graph showing the average standardized densitiesof EGF receptor mRNA hybridization in the striatal ridges, the non-ridgebody of the striatum, and the subependymal regions in all animals withstriatal ridges. The paired bars represent densities in striataipsilateral and contralateral to the treatment. Average hybridizationdensity was highest in the striatal ridge ipsilateral to the treatments.No striatal ridges ever appeared in the contralateral striata. TGFαstriatal infusions. Averages±S.E.M. (Student's t-test, paired foripsilateral-contralateral comparisons; P values, * p<0.005, ** p<0.001).

[0023]FIGS. 8A and 8B are photomicrographs of coronal sections throughthe striatum of an adult rat that received a nigral 6-OHDA lesion andTGFα infusion for fourteen days. In FIG. 8A, silver staining in thecaudate-putamen ipsilateral to the treatments reveals huge numbers ofcells in the dorsal portion of the ridge, many of which exhibit anelongated morphology and are oriented normal to the subependymal region.There is also an increase in the number of cells along the lateralventricle (lv). In the contralateral striatum (FIG. 8B), the cellularpopulation is not expanded, either in the striatum or along the lateralventricle.

[0024] FIGS. 9A-9D are photomicrographs of thionin-stained coronalsections of adult rat brain from animals that were lesioned with 6-OHDAand infused with TGFα for variable periods of time. In FIG. 9A, afterfour days of infusion, cellular expansion in the subependymal region isbarely detectable above background staining. In FIG. 9B, after six daysof infusion, the aggregation of thionin-stained cells near the lateralventricle is much more robust. In FIG. 9C, after nine days of infusion,a region of densely-stained cells appears slightly lateral to thesubependymal zone at the ventral end of the cellular expansion. In FIG.9D, after fourteen days of infusion, a dense, well-formed ridge isevident well into the body of the striatum.

[0025]FIGS. 10A and 10B are photomicrographs of coronal sections ofadult rat brain from animals that were lesioned with 6-OHDA and infusedwith TGFα for fourteen days. In FIG. 10A, nestin immunohistochemistryreveals an intense striatal ridge. In FIG. 10B, thionin staining of anear-adjacent section confirms the registry between the nestin-IR cellsand the striatal ridge.

[0026] FIGS. 11A-11C are photomicrographs of thionin stained coronalsections from adult rat brain from animals that were lesioned with6-OHDA and infused with TGFα, at varying distances from the lateralventricle, for fourteen days. In FIG. 11A, where the infusion cannulawas implanted in the far-lateral striatum, the ridge parallels thesubependymal zone and is less dense than that seen with a mid-striatalinfusion. In FIG. 11B, where the infusion cannula was implantedmid-striatum, the ridge is characteristically S-shaped, with the ventralportion extending far out into the ventral striatum. In FIG. 11C, wherethe infusion was immediately adjacent to the lateral ventricle, thestriatal ridge is L-shaped and generally exhibits very dense thioninstaining.

[0027]FIG. 12 is a bar graph depicting the maximum displacement of thestriatal ridge (from the lateral ventrical; in mm) in coronal sectionsof adult rat brain after nigral lesion with 6-OHDA and mid-striatalinfusion of TGFα for fourteen days. Animals treated according to“Schedule A” (left-most bar) received lesions first, followed by TGFαinfusions four to five weeks later. Animals treated according to“Schedule B” received lesions two days after the 14-day TGFα infusionbegan. Averages±S.E.M. (Student's t-test; P value, * p<0.01).

DETAILED DESCRIPTION

[0028] I. The Striatum and Nigrostriatal System

[0029] A. Anatomy, Connectivity and Neurochemistry

[0030] Within the brain, the striatum, pallidum, substantia nigra,ventral tegmental area (VTA), subthalamic nucleus, and amygdala arecollectively referred to as the basal ganglia. The striatum containsdorsal and ventral components, each of which is further subdivided intoadditional anatomical structures. In humans, the dorsal striatumconsists of the caudate nucleus and the putamen. The C-shaped caudatefollows the curve of the lateral ventricles. Its tail portions extendpast the ends of the inferior horns and joins the amygdala in eachtemporal lobe. The head of the caudate turns ventrally from the anteriorend of the anterior horns and fuses with the putamen. Although largelyanatomically distinct in the human brain, they are combined into acommon structure, the caudate-putamen or caudoputamen, in rodents.

[0031] The ventral striatum is comprised of the nucleus accumbens,olfactory tubercle, and the associated striatal cell bridges. Thepallidum includes the globus pallidus, entopeduncular nucleus,substantia nigra pars reticulata (SNr), and the ventral pallidum. Theentopeduncular nucleus and SNr have very similar afferent and efferentconnections. The ventral pallidum contains regions that have a mix ofconnections that are similar to both the globus pallidus andentopeduncular nucleus. The other part of the substantia nigra, the parscompacta (SNc), includes dopamine neurons that span the substantianigra-ventral tegmental area (SN-VTA), as well as dopamine cell clustersin the SNr. The circuitry of the basal ganglia is complex, but is verysimilar in both rats and humans (Fallon and Loughlin, Cerebral Cortex,E. G. Jones and A. Peters, Eds., Vol. 6, pp. 41-127, Plenum Press, NewYork, 1987; Alheid and Heimer, Progr. Brain Res. 107:461-484, 1996),making the rat a useful model for studying connections, neurochemistry,pharmacology, function, and clinical correlates of this system in themammalian brain.

[0032] The striatum, together with other nuclei of the basal ganglia,contributes to the regulation of movement and emotion. A number ofdiseases affecting the system or its innervation are associated withprofoundly debilitating motor impairment, often accompanied by affectivedisorders.

[0033] The caudate and putamen are the primary input nuclei of the basalganglia and receive major excitatory projections from the cerebralcortex and the centromedian and intralaminar nuclei of the thalamus.Corticostriatal afferents are glutamatergic. Afferents from the thalamusare also thought to be glutamatergic. The substantia nigra pars compacta(SNc) provides dense dopaminergic input to the striatum via thenigrostriatal pathway (for review of this system in the rat, see Fallonand Loughlin, The Rat Nervous System, G. Paxinos, Ed., pp. 215-237,Academic Press, San Diego, 1995). The ventral striatum and nucleusaccumbens receive the bulk of their dopaminergic innervation fromdopamine cells of the VTA in the ventromedial mesencephalon. Limbicafferents from the amygdala and serotonergic fibers from the midbrain orraphe also terminate in the ventral striatum.

[0034] The distribution of striatal afferents and their terminations arenot simply uniform representations of their regions of origin. Thestriatum is organized into patches or striosomes embedded in afunctionally and chemically distinct surrounding matrix. Thisorganization was originally demonstrated using histochemistry foracetylcholinesterase (AChE), which selectively stains the matrix(Graybiel and Ragsdale, Proc. Natl. Acad. Sci. USA, 75:5723-5726, 1978).Since then, enzyme histochemistry, immunocytochemistry, in situhybridization, receptor binding with radiolabeled ligands, anterogradedegeneration and other methods have been used to identify manyadditional markers that are differentially distributed in the twocompartments. Markers for the matrix include calbindin, somatostatin,and dopamine uptake ([³H]mazindol binding) sites (Gerfen, J. Comp.Neurol. 236:454-476, 1985; Voorn et al., J. Comp. Neurol. 289:189-201,1989). Striosomes can be identified by their higher relative abundanceof enkephalin, 5′-nucleotidase activity with nigrostriatal lesions,tyrosine hydroxylase, mu opoid receptor binding, and substance P(Graybiel et al., Neurosci. 6:377-397, 1981; Schoen and Graybiel, J.Comp. Neurol. 322:566-576, 1992). As might be expected, however, thereare interspecific and developmental variations in many of these markersand some are useful only in certain regions of the striatum.

[0035] The heterogeneity of chemical markers is further complicated bythe selective origin and termination of many striatal pathways in thepatch or matrix compartments. For instance, afferents from motor,cingulate, somatosensory and visual areas of cortex terminate in thematrix (Gerfen, Nature 311:461-464, 1984; Donoghue and Herkenham, BrainRes. 365:397-403, 1986). The bulk of the corticostriatal afferents fromdeep layer V and layer VI of limbic cortex terminate in the patcheswhile most from more superficial layer V and layers II and III provideinput to the matrix (Gerfen, Science 246:385-388, 1989). Afferents fromthe VTA and the dorsal tier of the SNc provide dopaminergic input to thematrix. The patches receive dopamine innervation from the ventral tierof the SNc and dopamine cell clusters in the SNr (Schoen and Graybiel,J. Comp. Neurol. 322:566-576, 1992). In the dorsal striatum, inputs fromnuclei in the medial division of the thalamus terminate in the patcheswhile afferents from the lateral division—including anterior andposterior intralaminar and rostral ventral tier nuclei—predominantlyinnervate matrix tissue (Ragsdale and Graybiel, J. Comp. Neurol.311:134-167, 1991). In addition, amygdalostriatal fibers originating inthe basolateral nucleus of the amygdala selectively innervate the patchcompartment (Ragsdale and Graybiel, J. Comp. Neurol. 269:506-522, 1988).

[0036] Striatal efferents are also differentially distributed withrespect to the patch-matrix organization. The striatonigral pathway, oneof the two major pathways originating in the striatum, has been shown tobe comprised of two distinct projections. Fibers arising from neurons inthe patch compartment terminate around dopamine neurons in the ventralSNc and in dopamine cell clusters in the SNr. Matrix neurons give riseto topographically-arranged projections to the SNr, includingnon-dopaminergic areas and dopamine neurons whose dendrites are locatedin the SNr (Gerfen, 1984, supra; Jiminez-Castallanos and Graybiel,Neurosci. 32:297-321, 1989).

[0037] The other major efferent projection, the striatopallidal pathway,projects to the globus pallidus. It has not been shown to be distributedwith respect to the patch-matrix organization; however, it isneurochemically distinct from the striatonigral system. The majority ofstriatopallidal fibers express enkephalin and not dynorphin or substanceP. In contrast, few striatonigral projections contain enkephalin, butmost express dynorphin and substance P (Gerfen and Young, Brain Res.460:161-167, 1988). In primates, the two systems also differ in theiranatomical regions of origin: striatopallidal efferents arise mainlyfrom the putamen while striatonigral efferents originate primarily inthe caudate (Parent et al., Brain Res. 303:385-390, 1984).

[0038] In addition to the heterogeneous distribution of striatalconnections, several morphologically and chemically distinct types ofneurons are found in the striatum (Albin et al., Trends Neurosci.12:366-375, 1989; Groves, Brain Res. Rev. 5:234-238, 1983). They aretraditionally classified as either spiny or aspiny based on theirdendritic morphology. There are two generally recognized types of spinyneurons in the striatum. They contain various combinations of GADA,substance P, enkephalin and dynorphin, but are predominantly GABAergic.The medium spiny neurons (spiny type I) are by far the most abundant,comprising 90-95% of all striatal neurons. They have smooth cell bodiesand dense accumulations of spines on the distant portions of theirdendrites. Their dendritic arborizations range to about 200 μm from thesomata. Medium spiny neurons are the principle terminal targets fordopaminergic neurons in the SNc, which form synapses predominantly onthe necks of the dendritic spines. Spiny type II neurons are muchlarger, with variable arbors extending up to about 600 μm from the soma.

[0039] Spiny neurons are the projection neurons of the striatum. Thosein the matrix containing GABA and substance P project predominantly tothe internal segment of the globus pallidus (GP_(i)) and the SNr. SpinyGABAergic matrix neurons containing enkephalin, on the other hand,innervate the external segment of the globus pallidus (GP_(e)). Spinyneurons in the patch compartment send the majority of their efferents tothe SNc (Albin et al., 1989, supra).

[0040] Striatal projection neurons of the two major efferent pathwayscan also be distinguished by their dopamine receptor subtypes. SubstanceP/dynorphin neurons projecting to the substantia nigra expresspredominantly D₁ dopamine receptors, while enkephalinergicstriatopallidal neurons express mainly D₂ receptors. Neither receptortype, however, is expressed exclusively in either projection (Besson etal., Neurosci. 26:101-119, 1989; Gerfen et al., Science 250:1429-1432,1990).

[0041] Three recognized types of aspiny neurons make up the populationof striatal interneurons (Groves, 1983, supra; Carpenter, In: Core Textof Neurosciences, pp. 325-360, Williams and Wilkins, Baltimore, 1991).Together, they make up 10% or less of the total number of neurons of thestriatum. Aspiny type I neurons are the most common of the three andhave smooth dendrites in arbors slightly smaller than those of mediumspiny neurons. They are largely GABAergic, but many contain somatostatinand neuropeptide Y. Aspiny type II neurons are distinguished by theirlarge cell bodies and AChE and choline acetyltransferase (CHAT)staining. This cell type forms symmetric synapses with medium spinyneurons. Medium aspiny type III neurons are the least well characterizedbut are thought to contain GABA. There are probably additionalchemically-defined and connectionally-defined subsets of these classesof neurons beyond the ones already identified.

[0042] B. Topography and Development

[0043] Experiments with anterograde and retrograde tracers in striatalprojections of the mesencephalic dopamine system revealed precisetopographies in adult rodents (Fallon and Moore, J. Comp. Neurol.180:545-580, 1978). The dorsal striatum receives dopaminergicinnervation from neurons in the ventral and intermediate SN and VTA. Theventral striatum and nucleus accumbens receive dopaminergic input fromthe dorsal VTA and intermediate SN (Fallon, Ann. NY Acad. Sci. 537:1-9,1988).

[0044] Neurogenetic gradients in the developing system parallel thetopographic arrangements of projections in the mature system. Thedorsolateral portion of the SN is the earliest produced in the embryo,before embryonic day 15 (E15) in the rat (Altman and Bayer, J. Comp.Neurol. 198:677-716, 1981). Projections from this region innervate thelateral and ventral regions of the striatum (Carter and Fibiger,Neurosci. 2:569-576, 1977; Veening et al., Neurosci. 5:1253-1268, 1980),which are also the earliest striatal areas generated (Bayer, Neurosci.4:251-271, 1984). As the striatum is populated with younger neurons in aventrolateral-to-dorsomedial gradient, afferents arrive from moreventromedial (and later-produced) portions of the SN (generated afterE15) to innervate these later-produced striatal areas. Thus, theyoungest (ventromedial) nigral dopamine neurons innervate the youngest(dorsomedial) striatal neurons, and older (dorsolateral) nigral dopamineneurons innervate older (ventrolateral) striatal neurons. This patternis repeated in the GABAergic striatonigral projections as well (Bunneyand Aghajanian, Brain Res. 117:423-435, 1976).

[0045] The neurons of the striatum are derived from a neuroepitheliumsurrounding the lateral ventricles in the prenatal and early postnatalbrain. A ventricular zone initially lines the ventricles, later joinedby the subventricular (or subependymal) zone just deep to it. As thebrain matures, the ventricular zone disappears, but the subependymalzone persists as a thin layer of cells. This zone contains neural stemcells and progenitor cells that migrate along a defined and restrictedpath to replenish the labile interneuronal cell population of theolfactory bulb (Luskin, Neuron 11:173-189, 1993; Lois andAlvarez-Buylla, Science 264:1145-1148, 1994).

[0046] C. Pathology

[0047] The striatum and its dopaminergic innervation are vulnerable to anumber of conditions including several neurodegenerative diseases (Albinet al., 1989, supra) such as Huntington's disease (HD) and Parkinson'sdisease (PD). HD is an autosomal dominant hereditary disease (chromosome4) characterized by progressive degeneration of the striatum. It isassociated with involuntary choreathetotic movements of the limbs andface and disruptions of voluntary movement (for review, see Purdon etal., J. Psychiatr. Neurosci. 16:359-367, 1994). Medium spiny GABAergicneurons in the matrix compartment are most affected, especially theGABA/enkephalinergic neurons projecting to the GP_(e) (Albin et al.,1989, supra). Large aspiny cholinergic interneurons and small aspinyinterneurons containing somatostatin, neuropeptide Y andNADPH-diaphorase, also found in the matrix compartment, are relativelyspared (Ferrante et al., Science 230:561-563, 1985; Reiner et al., Proc.Natl. Acad. Sci. USA 85:5733-5737, 1988). In more advanced stages of HD,however, neuronal degeneration includes all types of striatal neuronsand extends to other nuclei of the basal ganglia, the cerebral cortex,hypothalamus, and cerebellum.

[0048] Parkinson's Disease (PD) is characterized by resting tremor,rigidity, inability to initiate movement (akinesia) and slowness ofmovement (bradykinesia) (Marsden, Lancet 335:948-952, 1990). The motordeficits are associated with progressive degeneration of thedopaminergic nigrostriatal pathway and, to various extents, loss ofdopaminergic innervation to the nucleus accumbens and degeneration ofnoradrenergic cells of the locus ceruleus and serotonergic neurons ofthe raphe (Javoy-Agid et al., Adv. Neurol. 40:189-198, 1984; Agid,Lancet 337:1321-1327, 1991). Up to 80% of nigral-dopamine neurons can belost before significant motor deficits are manifest.

[0049] One of the major strategies for using peptides known asneurotrophic factors as therapeutic agents in the treatment ofneurodegenerative diseases is to arrest the degenerative process andenhance the function of remaining cells. The studies presented in theExamples below will illustrate how the present invention expands the useof neurotrophic factors beyond that which has been previously suggested.

[0050] II. Treatment of Neurological Deficits

[0051] As demonstrated by the examples below, neural precursors in theadult forebrain subependymal zone can be stimulated to proliferate andmigrate en masse into the central nervous system (CNS) (e.g., into thestriatum) in response to an infusion of a polypeptide that binds the EGFreceptor (e.g., TGFα; the term “polypeptide” as used herein refers toany chain of amino acid residues, regardless of length orpost-translational modification). Furthermore, one can direct migrationof the proliferating cells as a dense ridge. As described below,directed migration can be accomplished in a variety of ways. Forexample, it is facilitated by denervation of the target region (whichcan be achieved by neurochemical or mechanical forces), by applicationof a polypeptide growth factor (e.g., TGFβ, which increases the regionalexpression of cell adhesion molecules such as fibronectin and laminin),and by contacting the cells along a desired migratory path with acompound that inhibits a naturally occurring signal that would otherwiseinhibit migration (i.e., creating a permissive microenvironment byinhibiting an inhibitor).

[0052] Moreover, the shape of the migratory ridge can be controlled byvarying the location of the infusion (e.g., by altering the placement ofthe infusion cannula or of a biodegradable capsule containing the activecompound(s) of the invention). Similarly, the number of cells within theridge can be controlled by varying the dosage of the active compound(s)(e.g., the dosage of TGFα) or the distance at which it is releasedrelative to the population of neural precursor cells in the subependymalregion. As described below (and as illustrated in FIGS. 10A, 10B, 11A,11B, and 11C), when animals received mid-striatal or medial striatalinfusions, the migrating cells stopped before they reached the infusioncannulae, which resulted in S-shaped or L-shaped ridges. Thus, it ispossible not only to facilitate the cells' migration, but to controlwhere that migration ends. Adjusting the dose and the location ofrelease of the growth factor (and perhaps other compounds) may allowrestriction of the area affected to a relatively limited target region.

[0053] The proliferation and migration of neural precursor cells in theadult mammalian brain are distinct events that can be controlledseparately. Intracerebroventricular (ICV) or intrastriatal infusions ofTGFα or EGF without deafferentation can induce proliferation, butdegenerating, damaged (e.g., by deafferentation or other injury), orotherwise abnormal (i.e., malfunctioning) cells must be present tofacilitate migration, at least on a scale that is large enough to impactrecovery from an associated neurological deficit. As described furtherbelow, one can mimic the facilitating effect of degenerating, damaged,or otherwise abnormal cells with pharmacological agents. For instance,one can stimulate transient expression of cell adhesion molecules in thestriatum by administering an inductive compound. For example,fibronectin is strongly upregulated by transforming growth factor beta(TGFβ) in cultures of cerebellar astrocytes (Baghdassarian et al., Glia7:193-202, 1993). In transgenic mice overexpressing TGFβ, fibronectinand laminin are also strongly increased in the CNS over normal levels(Wyss-Coray et al., Am. J. Pathol. 147:53-67, 1995). Thus, directingmigration with any compound that stimulates the expression ofextracellular matrix molecules or cell adhesion molecules, particularlyalong the desired path of migration, is considered within the scope ofthe present invention.

[0054] Forebrain neural stem cells, which give rise to the migratingprogenitors, are believed to remain in place along the ventricular wall(Morshead et al., Neuron 13:1071-1082, 1994). In the experimentsdescribed below, a region of intense EGF receptor hybridizationpersisted along the lateral ventricle after the migratory ridge hadmoved into the striatum. In addition, elongated cells were always foundbetween the ridge and the lateral ventricle. Thus, despite the masscellular migration away from the subependymal zone, the stem cellsthemselves likely were not part of the migrating ridge. These neuralstem cells would provide a renewable source of new neurons and glia.Therefore, multiple waves of neural progenitor cells can be stimulatedto migrate into regions of the brain that are injured, or that havedegenerated or that otherwise contribute to a neurological deficit. Thepersistence of these cells also suggests that the normal function ofstem cells in the adult forebrain—presently thought to provide newneurons for the olfactory bulbs—should not be irreversibly disrupted bythe treatments.

[0055] Abundant striatal expression of TGFα (and its mRNA) and a lack ofdopaminergic innervation also characterize the early developing striatum(Weickert and Blum, Devel. Brain Res. 86:203-216, 1995; Bayer, Intl. J.Devel. Neurosci. 2:163-175, 1984). Similarly, the increased EGF receptormRNA expression in the subependymal region in TGFα-infused animalsmimics the abundant EGF receptor mRNA hybridization observed in theperiventricular neuroepithelium in the developing brain (Seroogy et al.,Neuroreport 6:105-108, 1994; Seroogy et al., Brain Res. 670:157-164,1995). Messenger RNAs encoding forms of fibronectin, and its receptor,and other cell adhesion molecules, which may facilitate the migration ofneural precursors, are also developmentally regulated (Pesheva et al.,J. Neurosci. Res. 20:420-430, 1988; Prieto et al., J. Cell Biol.111:685-698, 1990; Pagani et al., J. Cell Biol. 113:1223-1229, 1991;Linnemann et al., Int. J. Devel. Neurosci. 11:71-81, 1993). Thus, oneway to conceptualize the effects observed in the TGFα-infused and 6-OHDAlesioned animals in the present studies is as a selective recapitulationof embryonic neurogenesis. That is, neural stem cells in the adultmammalian brain may respond to proliferation signals and their progenymay respond to migration signals as they do in the developing animal.

[0056] Neural stem cells have recently been found in subependymathroughout the adult rodent CNS (Weiss, Soc. Neurosci. Abstr. 25:101,1995; Ray et al., Soc. Neurosci. Abstr. 22:394.5, 1996) and in thesubependyma of the adult human forebrain (Kirschenbaum et al., CerebralCortex 4:576-589, 1994). According to the methods described herein,these cells can be manipulated to provide a source of new neurons fordiseased, injured, or otherwise damaged or malfunctioning CNS neurons indiverse regions of the brain and spinal cord.

[0057] A. Advantages of the Present Invention

[0058] As described further below, one of the techniques proposed fortreating a neurological deficit involves removing neural precursors froma patient who has such a deficit and growing those cells in culture togenerate large numbers of neural progenitors. The cells may then bere-implanted into the same patient using techniques known to those ofordinary skill in the art (e.g., see Stein et al., In: Brain Repair, pp.87-103, Oxford University Press, New York, 1995, or Leavitt et al., Soc.Neurosci. Abstr. 22:505, 1996). Clearly, this technique is advantageousto those presently in use that require embryonic cells from abortedfetuses; it avoids altogether the ethical issues raised by the need touse aborted fetuses as tissue donors. In addition, it is more likely tosucceed because it will not stimulate the immune response that isresponsible for a high incidence of transplant rejection.

[0059] Stimulating proliferation and migration of neural precursors invivo has additional advantages; in vivo stimulation reduces the extentand possibly the number of invasive neurosurgical procedures. No stemcell excision surgery would be performed and multiple plugs oftransplanted cells, which are typically required with embryonic orcultured cell grafts, would not be necessary. Further, there would be nomassive die-off of undifferentiated neural progenitor cells due to thetransplantation procedure. Typically, with human fetal dopaminergic cellgrafts, 90% to over 99% of the implanted cells die before they becomeestablished in the host brain (Freed et al., Soc. Neurosci. Abstr.22:481.3, 1996).

[0060] Another advantage provided by the present invention is thatneural progenitor cells would not be isolated from the host brain byscar tissue. Plugs of transplanted cells become encapsulated within anenvelope of glyotic scar tissue and reactive astrocytes. In addition tothe physical barrier of the dense glyotic tissue, reactive astocyteswithin the scar tissue release factors which inhibit neurite outgrowth(McKeon et al., 1995). Neural progenitors created in vivo are notisolated from the rest of the brain by scar tissue. The outgrowth oftheir neurites, therefore, would not be inhibited by a massiveproliferation of reactive astrocytes. The directed migration providedfor herein therefore allows selective repopulation (which may vary inextent) of specific injured regions of the CNS with large numbers of newcells without disturbing undamaged areas.

[0061] The techniques presented here also represent an advance over thesingle previous study of forebrain neural stem cells stimulated in vivo.In that study, adult rats received ICV infusions of EGF for six days andwere followed for up to seven weeks post-infusion (Craig et al., J.Neurosci. 16:2649-2658, 1996). In the present study, TGFα was infusedfor fourteen days and followed for up to three months following the endof infusion. This difference is critical in that only in the presentstudy did the cells of the periventricular expansion migrate en masseinto the overlying striatum. The directed mass migration of neuralprogenitors into a selected target area represents a much preferredmethod to repopulate degenerated brain regions with new neurons.

[0062] One area of intense recent interest is the manipulation of neuralstem cell differentiation. Both the final location and the neurochemicalphenotypes of the cells once they have differentiated are of primaryimportance and are discussed further below.

[0063] When neural precursor cells were removed from adult rodent brainsand differentiated in vitro, cells immunochemically identified asastrocytes, oligodendrocytes and neurons are seen (Reynolds and Weiss,Science 255:1707-1710, 1992; Reynolds et al., J. Neurosci. 12:4565-4574,1992; Lois and Alvarez-Buylla, Proc. Natl. Acad. Sci. USA 90:2074-2077,1993). Many of the cells identified as neurons were also immunoreactivefor GABA and substance P, neurochemical markers for two cell typesnormally found in the striatum. Precursor cells explanted from the adulthuman brain also expressed neuronal markers and displayedelectrophysiological properties associated with neurons (Kirschenbaum etal., Cerebral Cortex 4:576-589, 1994).

[0064] These experiments suggest that when cells of the striatal ridgespontaneously differentiate in vivo, many of them will become cells withphenotypes typical of striatal neurons. Some recent data suggests thattheir phenotypes can be altered by exposure to different combinations ofneurotrophins (Lachyankar et al., Soc. Neurosci. Abstr. 22:394.7, 1996).Progenitor cells receiving different treatments expressed differentneurochemical immunomarkers once they differentiated, includingacetylcholinesterase, GABA, tyrosine hydroxylase (TH), and calbindin.The expression of TH was particularly interesting, since combinedproliferation, migration and directed differentiation into dopaminecells could provide a novel method to replace striatal dopamine lost inParkinson's disease (PD).

[0065] In PD patients, functionally significant numbers of newdopamine-producing striatal cells would aid in the reversal of motordeficits in a manner similar to transplants of aborted fetal midbraintissue. In patients with Huntington's disease (HD), neural precursorswould be stimulated to repopulate the striatum with new medium spinyGABAergic and other neurons lost to the disease. Some recent evidencefrom a different line of research indicates that reconstruction of thestriatopallidal pathway itself might be possible. Conditionallyimmortalized neural progenitor cells transplanted into the striatumdifferentiated and sent processes from the striatum to the globuspallidus (Lundberg et al., 1996).

[0066] B. Neurological Deficits Amenable to Treatment

[0067] Because the invention rests on the discovery that multipotentprecursor cells can be stimulated to divide and migrate through thebrain, it can be used to treat neurological deficits caused by a widevariety of diseases, disorders, and injuries. These insults include, butare not limited to, the following (others of skill in the art maycategorize differently the diseases and disorders listed below; howevercategorized, the neurological deficits with which they are associatedare amenable to treatment according to the methods of the presentinvention).

1. Degenerative Diseases

[0068] Degenerative diseases that can be treated according to themethods of the invention include Alzheimer's disease (AD), Parkinson'sdisease (PD), Huntington's disease (HD), Pick's disease, progressivesupranuclear palsy (PSP), striatonigral degeneration, cortico-basaldegeneration, childhood disintegrative disorder, olivopontocerebellaratrophy (OPCA; including a heritable form), Leigh's disease, infantilenecrotizing encephalomyelopathy, Hunter's disease,mucopolysaccharidosis, various leukodystrophies (such as Krabbe'sdisease, Pelizaeus-Merzbacher disease, and the like), amaurotic(familial) idiocy, Kuf's disease, Spielmayer-Vogt disease, Tay Sachsdisease, Batten disease, Jansky-Bielschowsky disease, Reye's disease,cerebral ataxia, chronic alcoholism, beriberi, Hallervorden-Spatzsyndrome, cerebellar degeneration, and the like.

2. Traumatic and Neurotoxic Injuries to the Central Nervous System

[0069] Traumatic and neurotoxic injuries that can be treated accordingto the methods of the invention include gunshot wounds, injuries causedby blunt force, injuries caused by penetration injuries (e.g., stabwounds), injuries caused in the course of a surgical procedure (e.g., toremove a tumor or abscess from the CNS or to treat epilepsy), poisoning(e.g., with MPTP or carbon monoxide), shaken-baby syndrome, adversereactions to medication (including idiosyncratic reactions), drugoverdose (e.g., from amphetamines), post-traumatic encephalopathy, andthe like.

3. Ischemia

[0070] Any disruption of blood flow or oxygen delivery to the nervoussystem can injure or kill cells, including neurons and glial cells,therein. These injuries can be treated according to the methods of thepresent invention and include injuries caused by a stroke (including aglobal stroke (as may result from cardiac arrest, arrhythmia, ormyocardial infarction) or a focal stroke (as may result from a thrombus,embolus, hemorrhage, or other arterial blockage)), anoxia, hypoxia,partial drowning, myoclonus, severe smoke inhalation, dystonias(including heritable dystonias), acquired hydrocephalus, and the like.

4. Developmental Disorders

[0071] Developmental disorders that can be treated according to themethods of the invention include schizophrenia, certain forms of severemental retardation, cerebral palsy (whether caused by infection, anoxia,premature birth, blood type incompatibility: etc. and whether manifestas blindness, deafness, retardation, motor skill deficit, etc.),congenital hydrocephalus, metabolic disorders affecting the CNS, severeautism, Down Syndrome, LHRH/hypothalamic disorder, spina bifida, and thelike.

5. Disorders Affecting Vision

[0072] Disorders affecting vision, particularly those caused by the lossor failure of retinal cells, can be treated according to the methods ofthe invention. These disorders include diabetic retinopathy, seriousretinal detachment, retinal damage associated with glaucoma, traumaticinjury to the retina, retinal vascular occlusion, macular degeneration,heritable retinal dystrophies, optic nerve atrophy, and other retinaldegenerative diseases.

6. Injuries and Diseases of the Spinal Cord

[0073] Injuries to or diseases affecting the spinal cord can also betreated according to the methods of the invention. Such injuries ordiseases include post-polio syndrome, amyotrophic lateral sclerosis,nonspecified spinal degeneration, traumatic injury (such as those causedby automobile or sporting accidents), including any injury that crushes,partially severs, completely severs, or otherwise adversely affects thefunction of cells in the spinal cord), injuries caused by surgery to thespinal cord (e.g., to remove a tumor), anterior horn cell disease,paralytic diseases, and the like.

7. Demyelinating or Autoimmune Disorders

[0074] Neurological deficits caused by demyelination or an autoimmuneresponse can be treated according to the methods of the invention. Suchdeficits can be caused by multiple sclerosis, possibly lupus, andothers.

8. Infectious or Inflammatory Diseases

[0075] Neurological deficits caused by an infection or inflammatorydisease can be treated according to the methods of the invention.Infections or inflammatory diseases that can cause treatable deficitsinclude Creutzfeldt-Jacob disease and other slow virus infectiousdiseases, AIDS encephalopathy, post-encephalitic Parkinsonism, viralencephalitis, bacterial meningitis and meningitis caused by otherorganisms, phlebitis and thrombophlebitis of intracranial venoussinuses, syphilitic Parkinsonism, tuberculosis of the CNS, and the like.

9. Miscellaneous

[0076] Those of ordinary skill in the art are well able to recognizeneurological deficits, regardless of their cause, and to apply themethods of the present invention to treat patients who have suchdeficits. In addition to the conditions listed above, which are amenableto treatment with the methods described herein, neurological deficitscan be caused by Lesch-Nyhan syndrome, myasthenia gravis, variousdementias, numerous parasitic diseases, epilepsy, and the like. Themethods of the invention can be readily applied to alleviateneurological deficits caused by these and other diseases, disorders, orinjuries.

[0077] C. Polypeptides That Bind the EGF Receptor

1. The EGF Family

[0078] Polypeptides in the EGF family appear, in some ways, unrelated.For example, TGFα and EGF have only 30% structural homology (Marquardtet al., Science 223:1079-1082, 1984). However, they display similarbinding kinetics for, and stimulate tyrosine-specific phosphorylationof, the M_(r) 180,000 EGF membrane receptor (Cohen et al., J. Biol.Chem. 255:4834-4842, 1980; Reynolds et al., Nature 292:259-262, 1981).The functional equivalence of the two growth factors is partlyattributed to the same relative positioning of six cysteine residues,represented by “C” in the concensus sequence: CX₇CX_(4,5)CX₁₀CXCX₈C.These conserved residues impose similar disulfide bond-mediatedstructural constraints and, thus, a related three-dimensional structure(Twardzik et al., Proc. Natl. Acad. Sci. USA 82:5300-5304, 1985). Thoseof ordinary skill in the art are well able to compare any given aminoacid sequence with the EGF-family concensus sequence to determinewhether a polypeptide is likely to be functionally equivalent to EGF(and, if so, useful in practicing the methods of the present invention).(see, e.g., Blomquist et al., Proc. Natl. Acad. Sci. USA 81:7363-7367,1984, for a description of a computer search that revealed a similarpattern of cysteine and glycine residues in EGF, TGFα, and the sequenceof a 19 kDa early protein of vaccinia virus).

[0079] In addition to EGF, TGFα, and vaccinia growth factor (VGF), theEGF family is known to include amphiregulin (AR), betacellulin (BTC),epiregulin (ER), heparin-binding EGF-like growth factor (HB-EGF),schwannoma-derived growth factor (SDGF), HUS 19878, myxomavirus growthfactor Shope fibroma virus growth factor, and teratocarcinoma-derivedgrowth factor-1 (TDGF-1; also known as Cripto-1 (CR-1).

2. Methods for Determining EGF Receptor Binding

[0080] Those of ordinary skill in the art are readily able to determinewhether any given polypeptide binds the EGF receptor. As used herein,the term “binds” refers to any specific interaction between apolypeptide and the EGF receptor that results in signal transductionsufficient to elicit a biological response, preferably a response thatcontributes to the reduction of a neurological deficit. Preferably, anygiven polypeptide useful in the methods of the present invention willbind the EGF receptor with an affinity that is equivalent to at least50%, more preferably at least 70%, and most preferably at least 90% ofthe binding affinity of EGF itself (see Twardzik et al., supra, for acomparison of the biological activity of VGF, TGFα, and EGF in EGFreceptor binding).

[0081] If guidance is required in performing an EGF receptor bindingassay, those of skill in the art can consult any one of numerouspublications describing a suitable procedure (the five publications onthis topic that follow are hereby incorporated by reference in theirentirety). For example, one could consult Cohen and Carpenter, Proc.Natl. Acad. Sci. USA 72:1317-1321, 1975) or, for a modification thereof,Twardzik et al., supra. Similarly, for review of the EGF receptor,including specific binding and sequence information, signalling, andreceptor topology, one may consult, for example, McInnes and Sykes(Biopolymers 43:339-366, 1997) Boonstra et al., (Cell Biol. Intl.19:413-430, 1995), or Gill (Mol. Reprod. Dev. 27:46-53, 1990).

[0082] D. Directed Migration

[0083] The examples below also provide evidence for successful directedmigration of neural precursor cells, particularly in the adult rodentforebrain. The immunohistochemical and other techniques employed in theworking examples below (and described at length therein), as well ascomparable techniques routinely performed by those of ordinary skill inthe art, can be used to characterize the effect of any infusion ofgrowth factor or any other stimulus applied to direct cellularmigration. Indeed, it is possible to trace the cells' migration in somedetail (i.e., the number of cells, their size, shape, and positionwithin the nervous system can be determined).

[0084] A variety of stimuli can be,applied to cells in vivo to directtheir migration en masse (the term “en masse,” when used herein todescribe cellular migration, refers to the movement of a population ofcells in substantially the same direction for a sufficient period oftime to be visualized as a mass (as, e.g., is apparent in FIGS. 9, 10,and 11)). Broadly, one can direct migration in one of two ways: (1) in aconducive manner, i.e., by applying a stimulus that positively attractsmigrating cells (such as a chemoattractant, neurotropic factor, or acompound (e.g., TGFβ) that increases the expression of a cell adhesionmolecule or extracellular matrix molecule (e.g., fibronectin, laminin,or a neural cell adhesion molecule)), or (2) in a permissive manner,i.e., by applying a stimulus that inhibits a signal that would otherwiseinhibit migrating cells.

[0085] The stimuli that direct migration include disruption of thetissue in the target area (which may be the site where cells have beendamaged, e.g., the striatum or substantia nigra, where dopaminergiccells are known to be lost in association with a number of debilitatingneurodegenerative diseases; the cerebral cortex, where neurons and gliaare lost following an ischemic episode caused by, e.g., a thrombus orembolus; or the spinal cord, where motor neurons are lost due to, e.g.,a traumatic injury; or may be any site where cells make abnormalconnections due to a developmental disorder). Alternatively, tissue maybe disrupted in or along a path extending from the source of the neuralprogenitor cells to the desired endpoint of their migration.

[0086] The tissue may be disrupted by physical force (e.g., ablating orexcising neurons, or severing one or more of the processes that extendfrom the neuronal cell bodies) or by applying a chemical substance suchas a toxin or neurotoxin (e.g., ricin or 6-OHDA), a corrosive chemical(e.g., an acidic or basic solution), a compound that induces apoptosis(see, e.g., Leavitt et al., Soc. Neurosci. Abstr. 22:505, 1996),.acompound that induces demyelination (see, e.g., Lachapelle et al., Soc.Neurosci. Abstr. 23:1689, 1997), or a compound capable of inhibiting theactivity of the cell, e.g., an antisense oligonucleotide (such as anoligonucleotide that inhibits transcription of the gene encoding thecell's primary neurotransmitter), an antibody, or a polypeptide. Manysuch compounds are known to those of ordinary skill in the art andinclude compounds that bind to, but fail to activate, a receptor on thecell surface, such as the metabotropic receptors normally bound byglutamate. For example, in the studies described below, the effect ofdopamine denervation with 6-OHDA (together with infusion of TGFα) oncellular migration is apparent.

[0087] Those of ordinary skill in the art are well able to directcellular migration by applying any of the chemical substances describedabove to a targeted area of the nervous system, particularly given theremarkably clear and accurate images of a patient's brain and spinalcord that can now be generated with, e.g., magnetic resonance imaging orcomputed tomographic scans.

[0088] Moreover, it is apparent from the studies described below thataltering one or more of the variables associated with application of acompound that directs migration (those variables including the nature,position, concentration, and duration of the application) can be alteredto direct more precisely the migratory path the cells follow and todefine the place at which they come to rest.

[0089] E. Differentiation Factors

[0090] While some neural precursor cells may spontaneouslydifferentiate, given enough time, substantially greater benefit can berealized by controlling when and where differentiation takes place;exerting such control allows one to limit neural “repopulation” (whichmay be partial or complete, so long as it is sufficient to reduce aneurological deficit) to areas of the CNS in need thereof. Accordingly,various stimuli can be administered before, during, or after contactingneural progenitor cells with a polypeptide that binds the EGF receptor.

[0091] Broadly, the stimuli inducing differentiation can be “general” or“directed.” A general differentiation stimulus is one that stimulates acell to differentiate as it naturally would and is applied whenever aneurological deficit can be reduced by stimulating a cell to express itsnatural phenotype. For example, it is known in the art that cells fromthe striatal subependymal zone differentiate into GABAergic neurons uponexposure to general differentiation signals. Thus, stimulating thesecells to differentiate by applying a general differentiation factorwould reduce the neurological deficits associated with Huntington'sDisease; in these patients, GABAergic medium spiny neurons are lostselectively.

[0092] Those of ordinary skill in the art are well able to apply theknown means of stimulating general differentiation to stimulatedifferentiation of the proliferating and migrating cells of the presentinvention. For example, contacting cells with a retinoblastoma proteinis known to cause them to exit the cell cycle, a requirement fordifferentiation (for a recent study, see, Slack et al., J. Cell Biol.140:1497-1509, 1998) and contacting cells with a cell cycle associatedkinase inhibitor, p21, can maintain cells in a post-mitotic (i.e.,differentiated) state (Berger et al., Soc. Neurosci. Abstr. 22:505,1996). Another stimulus that can be applied to stimulate differentiationin the context of the present methods is a cyclin D cell cycle regulator(Ouaghi et al., Soc. Neurosci. Abstr. 22:1706, 1996). Should one wish tostimulate differentiation of oligodendrocyte precursors, integrins maybe applied (see, e.g., Buttery et al., Soc. Neurosci. Abstr. 22:1723,1996). Brain-derived neurotrophic factor (BDNF) and retinoic acid (RA)are well known for their abilities to stimulate cellular differentiation(see, e.g., Ahmed et al., J. Neurosci. 15:5765-5778, 1995).

[0093] In the event neural precursor cells are cultured prior todirecting migration, they may be transplanted into an area of the brainthat is capable of influencing their differentiation. For example, ahigher percentage of transplanted neural precursor cells differentiatedinto neurons when placed near the subventricular zone (up to 35%)compared to those transplanted to more lateral sites (where only 0-8% ofthe cells differentiated) (Catapano and Macklis, Soc. Neurosci. Abstr.23:345, 1997). Similarly, transplanted cerebellar precursors expressmarkers for hippocampal neurons when they are transplanted into thehippocampus (Vicario-Abejon et al., J. Neurosci. 15:6351-6363, 1995).Accordingly, one of ordinary skill in the art will appreciate that, asan alternative to contacting the progeny of proliferating neuralprogenitor cells with a compound that stimulates their differentiation,the invention can be practiced by transplanting cells in or sufficientlynear a region of the brain that is capable of directing theirdifferentiation.

[0094] A directed differentiation stimulus is one that stimulates a cellto differentiate, but with a phenotype that is different from the one itwould naturally express. A directed differentiation factor would beapplied whenever a neurological deficit could be reduced by stimulatinga cell to express a non-natural phenotype. One such instance is in thecase of Parkinson's disease, where differentiation of striatal cellsinto dopamine-producing cells could substitute for the loss ofdopaminergic innervation from the substantia nigra. Similarly, one wouldaim to stimulate expression of a cholinergic phenotype in the septalregion, where cells are selectively lost in Alzheimer's Disease; inspinal motor neurons, which are lost in amyotropic lateral sclerosis andfollowing traumatic spinal injuries; and in oligodendrocytes, which arelost in demyelination disorders such as multiple sclerosis.

[0095] Factors from the GDNF/neurturin (TGFβ) family, which are derivedfrom a glial cell line, may induce differentiation in neural cells(which is, moreover, enhanced by RA) (Hishiki et al., Cancer Res.58:2158-2165), and GDNF stimulates motor neuron differentiation in ratventral mesencephalic cultures. BDNF and ciliary neurotrophic factor(CNTF) also promote motor neuron differentiation (their effects appearto be additive or synergistic to the effects of GDNF) (Zurn et al., J.Neurosci. Res. 44:133-141, 1996). In addition, motor neurondifferentiation can be induced by application of vitronectin, which isexpressed in the ventral region of the neural tube (Martinez-Morales etal., Development 124:5139-5147) and by the protein encoded by sonichedgehog (Tanabe et al., Curr. Biol. 5:651-658, 1995). A member of thesonic hedgehog family, Indian hedgehog, is expressed in developing andmature retina and promotes retinal progenitor proliferation andphotoreceptor development (Levine et al., J. Neurosci. 17:6277-6288,1997).

[0096] Cortical neural progenitors adopt a region-specific phenotypeinfluenced by EGF, TGFα and the type of substrate upon which they aregrown. EGF or TGFα doubled the percentage of limbic neurons derived fromnon-limbic-area precursors when they were plated on growthfactor-deficient Matrigel™ or collagen type IV (Ferri et al.,Development 121:1151-1160, 1995).

[0097] Insulin is known to affect differentiation of fetal neuron cellcultures, even more than IGF-1 (Abboud et al., Soc. Neurosci. Abstr.23:1425, 1997). Basic fibroblast growth factor (bFGF) and neurotrophinscan be used to direct the differentiation of hippocampal cells(Vicario-Abejon et al., Neuron 15:105-114, 1995).

[0098] In one embodiment, the methods of the invention can be applied torestore neural pathways that are lost to degenerative illness. Forexample, differentiated striatal GABAergic neurons can restorestriatopallidal projections upon their differentiation. Those ofordinary skill in the art are well able to recognize numerous neuralpathways that are amenable to reconstruction by the methods of thepresent invention.

[0099] F. Pharmaceutical Compositions

[0100] Polypeptides suitable for use in the present invention (i.e.,those that bind the EGF receptor or stimulate differentiation, alsoreferred to herein as “active compounds”) can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise one or more polypeptides and a“pharmaceutically acceptable carrier,” a term which is intended toinclude any and all solvents, dispersion media, coatings, antibacterialagents, antifungal agents, isotonic and absorption delaying agents, andthe like, that are compatible with pharmaceutical administration. Theuse of such media and agents is well known in the art. Except insofar asany conventional media or agent is incompatible with the activecompound, use thereof in the compositions is contemplated.

[0101] Those of ordinary skill in the art appreciate the need toformulate pharmaceutical compositions for their intended route ofadministration (which may include parenteral, e:g., intravenous,intradermal, or intramuscular injection; oral administration; or directapplication to the affected area). It is contemplated that the presentmethods will be carried out by applying polypeptides to neuralprecursors harvested from the brain and placed in culture or directly tothe precursor cells in vivo (by, e.g., infusion through an injectioncannula or shunt, or by implantation within a carrier, e.g., abiodegradable capsule) but other routes of administration, particularlyparenteral (preferably intravenous) administration, are also within thescope of the invention.

[0102] Solutions or suspensions useful in the pharmaceuticalcompositions of the present invention (e.g., in a composition containinga polypeptide that binds the EGF receptor and a compound that stimulatesthe differentiation of neural precursors) can include: sterile diluentssuch as water, normal saline, fixed oils, polyethylene glycols,glycerine, propylene glycol, or other synthetic solvents; antibacterialor antifungal agents such as benzyl alcohol, parabens (e.g., methylparabens), chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike; antioxidants such as ascorbic acid or sodium bisulfite; chelatingagents such as EDTA; buffers such as acetates, citrates, or phosphates;and agents for the adjustment of tonicity such as sodium chloride ordextrose. pH can be adjusted with acids or bases, such as hydrochloricacid or sodium hydroxide.

[0103] Pharmaceutical compositions suitable for injection includesterile aqueous solutions (where the active compound is water soluble)or dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, Cremophor EL™ (BASF; Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringability exists(proper fluidity can be maintained, for example, by using coatings suchas lecithin, by maintaining a certain particle size in the case ofdispersion, and by including surfactants). The composition must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi, as described above. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

[0104] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a polypeptide that binds the EGF receptor) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying, which yields a powder of the active compound plus anyadditional desired ingredient from a previously sterile-filteredsolution.

[0105] In one embodiment, the active compound is prepared with one ormore carriers that will protect it against rapid elimination from thebody, such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparing such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially, forexample, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

[0106] It is especially advantageous to formulate the compositions ofthe invention in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound, theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0107] In lieu of direct application of polypeptides that bind the EGFreceptor or stimulate cellular differentiation, nucleic acid moleculesencoding those polypeptides can be inserted into vectors and used asgene therapy vectors. Gene therapy vectors can be delivered to a subjectby, for example, intravenous injection, local administration (U.S. Pat.No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al.,Proc. Natl. Acad. Sci. USA 91:3054-3057, 1394). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded, for example, in thebrain or spinal cord. Alternatively, where the complete gene deliveryvector can be produced intact from recombinant cells, e.g., retroviralvectors, the pharmaceutical preparation can include one or more cellsthat produce the gene delivery system.

[0108] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0109] G. Treatment Regimes

[0110] By way of example (those of ordinary skill in the art are wellable to extrapolate from one model (be it an in vitro or in vivo model)to another, progressing toward optimal dosages for human patients), fora rodent brain, the infusions to stimulate proliferation of neuralprecursor cells were continued for a period of at least two weeks. Theresult being a dramatic increase in the numbers of undifferentiatedprogenitor cells along the adjacent ventricle. The continuous TGFαinfusion applied in the working examples below also supports radialcellular migration, but is not sufficient, by itself, to stimulate themassive radial migration observed in certain animals. The duration ofany treatment performed according to the methods described herein can bevaried according to the desired results. For example, in the workingexamples below, the cells greatly increased their numbers for more thana week prior to their mass migration away from the ventricle. Moreover,delayed migration was facilitated by denervation of the target region,either neurochemically or mechanically, and may also be facilitatedpharmacologically by the concurrent infusion of factor(s) such asanother neurotrophic factor, TGFβ, which increases the regionalexpression of cell adhesion molecules believed to underlie the radialmigration (as described above). The pattern of migration and the finallocation of the ridge of migrating cells can be controlled by alteringthe location of the infusion.

[0111] Those of ordinary skill in the art are well able to determine therequired dosage of a compound administered in the context of the presentinvention. Preferably, the dosage will range, whether infused orreleased from a time-release vesicle, from 1 to 100 ng/kg/day of theactive compound or ingredient (e.g., TGFα or any of the factorsdescribed above); more preferably, 1 to 50 ng/kg/day; and mostpreferably, 1 to 10 ng/kg/day will be administered.

EXAMPLES Example 1 Expression of TGFα and EGF Receptor mRNAS in theNormal Developing and Adult Nigrostriatal System

[0112] As reviewed in the Detailed Description, mRNAs encodingEGF-family neurotrophic factors are developmentally regulated in thenigrostriatal system. In the studies described below, the expression ofTGFα and EGF receptor mRNAs is examined in the normal developing andadult rodent system.

[0113] A. Animals and Tissue Preparation

[0114] Adult male adult and timed-pregnant female Sprague-Dawley rats(250-350 g) were obtained from Simonsen (Gilroy, Calif.). For theseexperiments and all others described herein, the animals were maintainedin a temperature and humidity controlled vivarium. Use of the animalsfor all of the experimental procedures employed was approved by theUniversity of California, Irvine, Animal Research Committee inaccordance with National Institutes of Health guidelines.

[0115] Newborn (P0), postnatal day 1 (P1), and P4 animals wereanesthetized by hypothermia and sacrificed by decapitation. P10, P21,and adult animals were sacrificed by decapitation. Their brains werequickly removed, frozen in isopentane at −20° C., and stored at −70° C.Coronal cryostat sections were cut at 20 μm and thaw-adhered toVectabond™ (Vector Labs, Inc.) coated slides in orderedanterior-to-posterior rows. The sections were postfixed with 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hour, rinsedin phosphate buffer and air dried. Sections were stored with desiccantat −20° C until processed.

[0116] B. Hybridization Probes

[0117] TGFα mRNA probes were generated from a 550 nucleotide XbaI/BamHIcDNA fragment from the 5′ end of rat TGFα and subcloned into pGEM 7Zf(Promega, Inc.). Antisense and sense probes were transcribed with SP6and T7 polymerases, respectively. Rat EGF receptor mRNA probes wereproduced from a 718 base pair BamHI/SphI insert from the 5′ end of thegene, in pGEM 7Zf. Probes for rat TH were created using the 1.2 kbBamHI/EcoRI fragment subcloned into pGEM 7Zf. Antisense subclones forEGF receptor and TH were transcribed with T7 polymerase. Sense subclonesfor EGF receptor and TH were transcribed with SP6 polymerase. All probeswere radiolabeled by transcription in the presence of [³⁵S]UTP (NENResearch Products, Inc.).

[0118] C. In situ Hybridization and Analysis

[0119] In situ nucleic acid hybridization was performed according to themethod described by Simmons et al., (J. Histotech. 12:1169-181, 1989)except that developing brains were treated with 0.0001% proteinase Ksolution and 0.05 M EDTA. Sections were hybridized overnight at 65° C.with sense or antisense probes at a concentration of 10⁷ cpm/ml.Adjacent sections from the same animals were hybridized to each of theprobes so that direct comparisons could be made of their anatomicaldistributions.

[0120] Slides from developing and adult animals were grouped togetherand apposed with ¹⁴C-labeled brain paste standards to autoradiographicBetaMax Hyperfilm (Amersham, Inc.) for six to seven days. Aftersuccessful development of the autoradiography film, the slides weredipped in Kodak NTB-2 emulsion and exposed for four weeks. Theautoradiographic sheet film and NTB-2 emulsion were developed with D-19developer and Rapid Fix (Kodak, Inc.). The brain sections were thencounterstained with thionin and coverslipped. Dipped and stainedsections were examined semiquantitatively and photographed under brightand dark field microscopy.

[0121] Expression of TGFα and EGF receptor mRNAs in the nigrostriatalsystem was traced through selected time points from early postnataldevelopment to adulthood. TGFα mRNA hybridization was found in abundancein the early postnatal striatum but was gradually reduced to near adultlevels by P21. Expression in the corpus callosum increased throughpostnatal development to levels comparable to those in the striatum.TGFα mRNA was not detected in significant abundance in the developing orthe adult substantia nigra.

[0122] Striatal EGF receptor mRNA peaked early in postnatal developmentand decreased again by P21. EGF receptor was highest in theneuroepithelia around the lateral ventricles, but was also found atmoderate levels in the body of the striatum. In the developing ventralmidbrain, EGF receptor mRNA was barely detectable in early postnatalbrains, but gradually increased to moderate levels by P21.

[0123] In adult animals, TGFα mRNA expression was moderate in thestriatum and low-to-moderate in the ventral striatum and nucleusaccumbens. EGF receptor mRNA hybridization was found at low levels inthe body of the striatum and nucleus accumbens with higher punctateexpression dispersed throughout. It persisted at moderate levels in theregions of striatum immediately bordering the lateral ventricles.

[0124] In the adult ventral midbrain, EGF receptor mRNA hybridizationwas found in the substantia nigra (SN), particularly the medial parscompacta, and the paranigral and parabranchial nuclei of the ventraltegmental area (VTA).

[0125] Previous studies indicated that TGFα and EGF receptor mRNAs arestrongly regulated during ontogeny of the nigrostriatal system and thattheir expression in the adult largely represents a continuation of thedevelopmental pattern (Lazar and Blum, J. Neurosci. 12:1688-1697, 1992;Weickert and Blum, Devel. Brain Res. 86:203-216, 1995). TGFα and EGFreceptor mRNA hybridization in the developing and adult animals closelyparalleled the findings of these earlier reports. The persistence oftheir expression in the adult striatum and midbrain is consistent with asupportive role in the mature nigrostriatal system.

[0126] The moderate EGF receptor mRNA expression in the adultsubependymal regions along the forebrain lateral ventricles suggests arole in the maintenance or function of cells in this region as well(Seroogy et al., Brain Res. 670:157-164, 1995; Weickert and Blum, 1995,supra). TGFα (or EGF) has been shown to support the survival anddifferentiation of “EGF-responsive” cells from this region when they areexplanted and grown in vitro (Reynolds and Weiss, Science 255:1707-1710,1992; Reynolds et al., J. Neurosci. 12:4565-4574, 1992). It may performa similar function in vivo during development.

Example 2 Modulation of TGFα and EGF Receptor mRNA Expression by6-hydroxydopamine Lesion and Striatal TGFα Infusion

[0127] In situ hybridization was used to determine whether nigrostriatalTGFα or EGF receptor mRNA was altered by intrastriatal infusion of TGFα.In addition, the influence of unilateral 6-OHDA lesions on receptorexpression in infused and uninfused animals was examined.

[0128] A. Treatment Groups

[0129] Adult male Sprague-Dawley rats weighing 250-300 grams wereobtained from Simonsen (Gilroy, Calif.) and assigned to one of fivetreatment groups: (1) striatal TGFα infusion, nigral 6-OHDA lesion(hereafter, “lesion”); (2) TGFα infusion, no lesion; (3) artificialcerebrospinal (aCSF) infusion, lesion; (4) aCSF infusion, no lesion; (5)no infusion, no lesion. Four to eight animals were used per experimentalgroup. The animals were monitored after each surgical procedure untilfully recovered and maintained at all other times in a temperature andhumidity controlled vivarium.

[0130] B. 6-hydroxydopamine Lesions

[0131] Rats were anesthetized with 8 mg xylazine and 100 mg ketamine perkilogram body weight. A chilled solution of 4.8 mg/ml 6-hydroxydopamineHCl (6-OHDA; Sigma Chemical Co.) in 0.9% saline with 0.01% ascorbic acidwas prepared immediately before injection. Using sterile technique, an 8μl volume was stereotaxically infected into the left substantia nigra(+3.7 A/P; +2.1 M/L; +2.0 D/V) at a rate of 1 μl/minute using interauralzero as a reference (Paxinos and Watson, The Rat Brain in StereotaxicCoordinates, Academic Press, San Diego, 1986). The success and extent of6-OHDA lesions were monitored by tyrosine hydroxylase (TH) mRNA in situhybridization in the midbrain. TH is the rate-limiting enzyme in thedopamine synthetic pathway and is a common marker for dopamine-producingneurons. One animal with an incomplete lesion (retaining significantnumbers of nigral TH-IR cells) was excluded from the study and is notincluded in the total number of animals.

[0132] C. Infusions

[0133] Osmotic minipumps (model 2002, Alzet, Inc.) were implanted fourto five weeks post-lesion. The minipumps were filled with approximately200 μl of either 0.05 μg/ml TGFα in artificial cerebrospinal fluid(aCSF) for experimental animals, or aCSF only for control animals, andincubated overnight at 37° C. prior to implantation. Followinganesthesia as above, and under sterile conditions, the 5 mm cannulaattached to the minipump (brain infusion kit, Alzet, Inc.) wasstereotaxically implanted into the left caudate-putamen (+1.2 A/P; +2.7M/L) using Bregma as a reference (Paxinos and Watson, 1986, supra) andfixed to the skull with carboxylate cement (FIG. 1). The minipump itselfwas placed subcutaneously in the interscapular region. The infusate wasdelivered directly into the striatum over a period of two weeks at arate of 0.5 μl/hour.

[0134] D. Tissue Preparation

[0135] At the end of the infusion period, animals were sacrificed bydecapitation. Their brains were quickly removed, frozen in isopentane at−20° C., and stored at −70° C. Coronal cryostat sections were cut at 20μm and thaw-adhered to Vectabond™ (Vector Labs, Inc.) coated slides inordered anterior-to-posterior rows. The sections were postfixed with 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for one hour, rinsedin phosphate buffer and air dried. Sections were-stored with desiccantat −20 C. until processed.

[0136] E. Hybridization Probes

[0137] TGFα mRNA probes were generated from a 550 nucleotide XbaI/BamHIcDNA fragment from the 5′ end of rat TGFα subcloned into pGEM 7Zf(Promega, Inc.). Antisense and sense probes were transcribed with SP6and T7 polymerases, respectively. Rat EGF receptor mRNA probes wereproduced from a 718 base pair BamHI/SphI insert from the 5′ end of thegene in pGEM 7Zf. Probes for rat TH were created using the 1.2 kbBamHI/EcoRI fragment subcloned into pGEM 7Zf. Antisense subclones forEGF receptor and TH were transcribed with T7 polymerase. Sense subclonesfor EGF receptor and TH were transcribed with SP6 polymerase. All probeswere radiolabeled by transcription in the presence of [³⁵S]UTP (NENResearch Products, Inc.).

[0138] F. In situ Hybridization

[0139] In situ nucleic acid hybridization was performed according to themethod described by Simmons et al. 1989, supra). Parallel sections fromexperimental and control animals were hybridized overnight at 65° C.with sense or antisense probes at a concentration of 10⁷ cpm/ml.Adjacent sections from the same animals were hybridized to each of theprobes so that a direct comparison could be made of their anatomicaldistributions.

[0140] Slides from experimental and control animals were groupedtogether and apposed with ¹⁴C-labeled brain paste standards toautoradiographic BetaMax Hyperfilm™ (Amersham, Inc.) for three to sevendays. After successful development of the autoradiography film, theslides were dipped in Kodak NTB-2 emulsion and exposed for four weeks.The autoradiographic sheet film and NTB-2 emulsion were developed withD-19 developer and Rapid Fix (Kodak, Inc.). The brain sections were thencounterstained with thionin and coverslips were applied.

[0141] G. Analysis and Quantitation

[0142] Dipped and stained sections were examined and photographed underbright and dark field microscopy. Autoradiograms were analyzedquantitatively using an MCID system (Microcomputer Imaging Device,Imaging Research, Inc.). Densitometry readings were sampled at multiplesites within each anatomical region of interest and averaged. Relativeconcentrations of TGFα and EGF receptor hybridization were thenestimated using a computer-generated third degree polynomial standardcurve constructed from the ¹⁴C brain paste standards. The estimatedvalues for each region in each treatment group were then averaged andtheir standard errors calculated. Brain regions ipsilateral to theexperimental treatments were compared to the corresponding contralateralregions in the same sections or to corresponding regions in controlbrains at approximately the same positions. Significance of thecomparisons was determined using the Student's t-test.

[0143] H. Normal Expression and Control Infusions

[0144] Expression of TH, TGFα and EGF receptor mRNAs in the striatum,the striatal subependymal region, and the SN in control animalsreceiving striatal infusions of aCSF was indistinguishable from that innormal animals (See Example 1 for normal developing and adultexpression). TH mRNA hybridization was prominent throughout the SN-VTA.TGFα mRNA was not detected in the SN. EGF receptor mRNA, however, wasprominent in the medial substantia nigra pars compacts (SNc), theparanigral, parabranchial, and interpeduncular nuclei of the VTA (FIG.2).

[0145] TGFα mRNA hybridization was expressed in the caudate-putamen andnucleus accumbens (NA), being slightly less dense in the NA. EGFreceptor hybridization was found at low levels throughout the body ofthe striatum and NA with higher punctate expression dispersed throughoutthe low level background, and at moderate levels in the proliferativeregions of striatum bordering the lateral ventricles.

[0146] I. Effects of 6-OHDA Lesions

[0147] Unilateral nigral 6-OHDA lesions reduced TGFα mRNA hybridizationin the ipsilateral striatum by 25%, but had no effect on contralateralTGFα mRNA hybridization (FIG. 5). Striatal and subependymal EGF receptorhybridization were unchanged in lesioned animals compared to normalanimals (FIG. 6). In the midbrain, 6-OHDA lesions abolished EGF receptorhybridization in the ipsilateral SN-VTA.

[0148] J. Effects of TGFα Infusions

[0149] In unlesioned animals receiving TGFα infusions, TGFα mRNAhybridization in the infused striatum was unchanged compared to thecontralateral striatum or striata from normal animals (FIG. 5). A few ofthe animals receiving infusions of either TGFα or aCSF displayed aslight increase of TGFα mRNA hybridization immediately around theinfusion cannula scar. TGFα mRNA hybridization in the substantia nigrawas not increased to detectable levels by the infusions.

[0150] Hybridization to EGF receptor mRNA was dramatically increased inthe ipsilateral subependymal region, but not in the rest of thestriatum, in all animals receiving TGFα infusions (FIG. 6). No changefrom normal was observed in EGF receptor hybridization in the SNc withTGFα infusion alone.

[0151] K. Combined TGFα Infusion and 6-OHDA Lesion

[0152] Striatal TGFα mRNA hybridization in animals receiving bothstriatal TGFα infusions and subsequent nigral 6-OHDA lesions wasindistinguishable from that in lesion-only animals (FIG. 5). Similarly,EGF receptor hybridization in the midbrains ofTGFα-infused/6-OHDA-lesioned animals was indistinguishable from that inlesion-only animals.

[0153] EGF receptor mRNA hybridization in the forebrain revealed ananomalous ridge of dense hybridization in the body of the ipsilateralstriatum in addition to the increased hybridization in the subependymalregion (FIGS. 4, 6, and 7). The ridge was found in five of the six ratsin the combined TGFα infusion/lesion group, but only in one of six ratsin the TGFαinfusion/nonlesion group. EGF receptor hybridization in thesurrounding striatum was unchanged.

[0154] L. Discussion

[0155] The results described above permit analysis of the modulatoryeffects of nigral 6-OHDA lesions or striatal infusions of TGFα, or both,on the expression of mRNAs encoding TGFα and the EGF receptor in theadult rodent nigrostriatal system. The results clearly demonstratedchanges in expression associated with each treatment individually and aunique pattern of striatal expression when the two treatments werecombined.

1. Effects of 6-OHDA Lesions

[0156] Midbrain lesions with 6-OHDA reduced TGFα mRNA hybridization inthe striatum by 25% at several weeks post-lesion. If TGFα peptide levelsparallel expression of TGFα mRNA in this system, the decrease in TGFαmRNA may be one aspect of the rodent lesion model that is not similar toidiopathic human PD: TGFα is greatly increased in the striata of PDpatients (Mogi et al., Neurosci. Lett. 180:147-150, 1994). TGFα has beenshown to enhance a number of measure of dopamine neuron function invitro (Alexi and Hefti, Neurosci. 55:903-918, 1993). The increase ofTGFα (and EGF and other trophic factors) may therefore reflect aresponse to the continuing degeneration of dopamine neurons and theirstriatal efferents and may contribute to the capacity of remainingmidbrain dopamine cells to compensate for the lost striatal dopaminergicinnervation (Mogi et al., 1994, supra). Thus, a partial- or perhaps achronic-injury model might better represent this aspect of human PD.

[0157] The difference in the time course of the loss of dopamine cellsmay also help explain the apparent discrepancy between the 6-OHDA rodentmodel and human PD. In the rat model, midbrain dopamine neurons arekilled relatively quickly by a single injection of neurotoxin. Thechronic, progressive degeneration of mesencephalic dopamine neurons inhuman PD occurs over many years. In the present study, the animals weresacrificed well after midbrain dopamine cells had degenerated. There mayhave been early changes in striatal TGFα mRNA expression in theseanimals that would not be apparent in our experiments. It would be ofinterest to determine how TGFα mRNA expression varies in the rat modelover shorter periods post-lesion, while dopamine neurons are in theprocess of degenerating.

[0158] Moderate TGFα mRNA expression in the caudate-putamen isconsistent with its putative role as a target-derived growth factor formidbrain dopamine neurons. The underlying cause of its decrease in theipsilateral striatum after midbrain neurotoxic lesion is unclear.Dopamine receptor binding has been shown to influence TGFα mRNAexpression in the hypothalamus (Borgundvaag et al., Endocrinol.130:3453-3458, 1992), but no such interaction has yet been demonstratedin the striatum. TGFα mRNA is expressed in subpopulations of neurons andglia in the normal adult rodent striatum (Seroogy et al., J. Neurochem.60:1777-1782, 1993). Dopamine denervation of the striatum couldpotentially have influenced TGFα mRNA expression in postsynapticneurons, astrocytes or both.

[0159] Contralateral striatal TGFα mRNA expression was not significantlyaltered by 6-OHDA lesion. This finding, too, is consistent with adopamine denervation-mediated decrease in TGFα mRNA expression. Only afew percent of mesostriatal dopaminergic projections are contralateral(Loughlin and Fallon, Neurosci. Lett. 32:11-16, 1982), thus anycontralateral regulatory effects resulting from the lesion would beexpected to be minor compared to ipsilateral effects.

[0160] EGF receptor mRNA hybridization in the DA-denervated striatum didnot differ significantly from that in the contralateral CP or inunlesioned control striata. Again, there may have been early changes inexpression due to the midbrain lesion or implantation of the infusioncannula that were not manifest at several weeks postlesion or two weekspost-implantation. The abolition of EGF receptor mRNA hybridization inthe lesioned SNc confirms a similar observation after 6-OHDA lesion ofthe medial forebrain bundle (Seroogy et al., Neuroreport 6:105-108,1994). This lesion-induced decrease was previously cited as evidence ofEGF receptor expression by nigral dopamine neurons (Seroogy et al.,1994, supra), but the possibility remains of nigral glial production ofEGF receptor mRNA that is subject to regulation by injury or death ofnearby nigral dopamine neurons. Interestingly, EGF receptor binding inthe midbrains of postmortem PD patients is unchanged from normals(Villares et al., Brain Res. 628:72-76, 1993). Thus, the loss of EGFreceptor mRNA expression after lesion represents another differencebetween the rodent lesion model and human PD. As with TGFα expression inthe striatum, a partial-lesion model may better mimic in a rat brain thechanges seen in a Parkinsonian human brain.

2. Effects of TGFα Infusions

[0161] In unlesioned animals, infusion of TGFα or aCSF did notsignificantly alter TGFα mRNA hybridization from normal levels in themidbrain or striatum. Despite reports of autostimulation of TGFαexpression in other tissues or cell types (Coffey et al., Nature328:817-820, 1987; Barnard et al., J. Biol. Chem. 269:22817-22822,1994), results from the present study do not provide evidence for suchactivity in this system. The autostimulatory effects in those earlierstudies were produced on the order of a few hours. Brain tissue in thepresent study was obtained after continuous exposure to the growthfactor over a period of two weeks. Thus, an early upregulation near thebeginning of the infusion that later subsided would not be evident.

[0162] As with TGFα and EGF receptor transcripts in lesion-only animals,it would be of interest to examine the time course of modulation at timepoints earlier after onset of the experimental treatment. The slightincrease in TGFα mRNA seen in a few animals immediately around theinfusion scar was found in both TGFα- and aCSF-infused striata. Thus, itis probably attributable to continued mechanical injury and gliosiscaused by the cannula itself, and not to the infusate.

[0163] TGFα infusions dramatically increased EGF receptor mRNAhybridization in the ipsilateral subependymal zone but not in the restof the striatum. In other tissues, EGF receptor mRNA can be modulated byseveral chemical and mechanical means. EGF peptide increased EGFreceptor mRNA in numerous mammalian cell types in vitro (Earp et al., J.Biol. Chem. 261:4777-4780, 1986; Kesavan et al., Oncogene 5:483-488,1990). Retinoic acid caused a similar increase in normal rodentfibroblasts (Thompson and Rosner, J. Biol. Chem. 264:3230-3234, 1989)and in a transformed rat liver cell line (Raymond et al., Cell. GrowthDiff. 1:393-399, 1990). Exposure to cyclohexamide, by itself or withEGF, stimulated an increase in cultured human cytotrophoblasts andstabilized EGF receptor transcripts, thus providing a mechanism otherthan enhanced transcription to increase total abundance of EGF receptormRNA (Kesavan et al., 1990, supra).

[0164] Transection of the sciatic nerve in rats brought about a gradedincrease in EGF receptor mRNA in the severed ends (Toma et al., J.Neurosci. 12:2504-2515, 1992). Treatment with protamine increased¹²⁵I-EGF binding and cell surface receptor number in mouse and humancell lines in vitro (Lokeshwar et al., J. Biol. Chem. 264:19318-19326,1989). TGFα may mimic these actions and increase the production and/orlongevity of EGF receptor transcripts in extant subependymal cells. Itmay also stimulate transcription in cells that do not normally expressappreciable amounts of EGF receptor mRNA. Both of these effects could bereadily investigated further in vivo with routine techniques known tothose of ordinary skill in the art.

[0165] An additional possibility is that TGFα is stimulatingproliferation of subependymal cells and increasing the total numbers ofcells expressing EGF receptor mRNA. EGF and TGFα are potent mitogens forcultured “EGF responsive” cells explanted from the subependymal region(Reynolds and Weiss, 1992, Science 255:1707-1710). The strong inductiveeffect striatal TGFα infusion had on subependymal EGF receptor mRNAhybridization may indicate that proliferative cells in the intact brainrespond similarly to these cell in vivo.

3. Combined TGFα Infusion and 6-OHDA Lesion

[0166] In animals receiving combined lesions and TGFα infusions,striatal TGFα mRNA hybridization was indistinguishable from that inanimals receiving combined lesions and aCSF infusions. Although TGFα haspotent autostimulatory effects in other tissues, it did notsignificantly alter the reduction of striatal TGFα mRNA hybridization inthe present study. The 6-OHDA-mediated loss of mesencephalic EGFreceptor mRNA was similarly unaffected by TGFα infusion. In the lattercase, the midbrain lesions were performed, and ipsilateral dopaminecells destroyed, weeks prior to the start of the infusion. Thus, thegrowth factor would not have had an opportunity to prevent theirelimination.

[0167] There is some evidence that the dopamine cells themselves expressEGF receptor mRNA (Seroogy et al., 1994, supra) and that TGFα canmoderate the loss of markers for striatal dopaminergic innervation ifadministered concurrently with the neurotoxin. Therefore, the timeinterval between the neurotoxic lesions and the administration of TGFαmay explain why TGFα infusions had no impact on the abolition ofmidbrain EGF receptor mRNA.

[0168] In human Parkinsonian brains, mesencephalic EGF receptor bindingis unchanged from that seen in the normal brain (Villares et al., 1993,supra). The huge increases in striatal TGFα (and other neurotrophicfactors) with PD (Mogi et al., 1994, supra) may mask a reduction in thenumber of EGF receptor expressing dopamine cells by increasing thelevels of expression in the remaining neurons. On the other hand, invitro experiments suggest that many of the trophic effects of TGFα onmesencephalic dopamine neurons are mediated, at least partially, throughglia (Alexi and Hefti, 1993, supra). TGFα may therefore act throughparacrine (direct) and sequential (indirect) modes of transport toinfluence dopamine neurons.

[0169] The pattern of EGF receptor mRNA hybridization in thesubependymal zone of TGFα-lesioned animals was similar to that seen inTGFα-nonlesioned animals. The most striking feature in the ipsilateralstriata of these animals was a dense ridge of hybridization well out ofthe body of the striatum, more intense even that the enhancedhybridization in the subependymal zone. The ridge did not correspond toany known anatomical feature and was not evident with the TGFα or THprobes. EGF receptor mRNA hybridization in the non-ridge striatum wasthe same as in the striata of all other groups.

[0170] The neurotoxic damage from the 6-OHDA lesions and the mechanicalinjury from implantation of the infusion cannula may have stimulatedproliferation and activation of glial cells. Previous studies havedemonstrated gliosis and increased astrocytic EGF receptor expression asa result of injury (Nieto-Sampedro et al., Neurosci. Lett. 91:276-282,1988; Fernaud-Espinoza et al., Glia 8:277-291, 1993). Further, TGFα mayplay a role in the reactivity of astrocytes (Junier et al., J. Neurosci.14:4206-4216, 1994).

[0171] Another possibility is that proliferative cells of thesubependymal region were drawn away from the ventricle and into theoverlying striatum by the combined growth factor infusion and midbrainlesion. TGFα is a potent chemoattractant for diverse cell types (Renekeret al., Development 121:1669-1680, 1995), but by itself was notsufficient in most animals to stimulate formation of the ridge.Formation of the cellular ridge may have been facilitated by themidbrain lesions. The origin and identity of these cells will beexamined in the following example.

Example 3 Characterization of the Striatal Ridge

[0172] As described above, striatal infusions of TGFα, when combinedwith nigral 6-OHDA lesions, induce the formation of a dense ridge ofcells in the body of the striatum that abundantly expresses EGF receptormRNA but no more TGFα than the surrounding tissue. The ridge wascomprised of a mass of densely packed cells, allowing its cleardetection using simple thionin staining. The identity of the anomalousstriatal ridge was not apparent, but three possibilities wereconsidered.

[0173] Gliosis in response to injury is a feature of both traumatic andneurotoxic damage to brain tissue. Typically, both types of brain injurystimulate astrocytosis and infiltration of injured tissue by astrocytesand microglia (Fernaud-Espinoza et al., Glia 8:277-291, 1993).Astrocytes have been shown to express EGF receptor immunoreactivity,particularly in response to brain injury (Gomez-Pinilla et al., BrainRes. 438:385-390, 1988; Nieto-Sampedro et al., Neurosci. Lett.91:276-282, 1988). In addition, TGF itself stimulates the proliferationof astrocytes (Alexi and Hefti, Neurosci. 55:903-918, 1993). Therefore,the possibility that the striatal ridge was a mass of glial cellsresponding to the combined neurotoxic and mechanical damage and infusionof the growth factor was considered.

[0174] A second potential source for the ridge was investigated that wasrelated to the distinctive anatomy of the rodent striatum. The ridge didnot correspond to any previously identified anatomical feature. Inrodents, the caudate and putamen are not anatomically distinctstructures. No anatomical or neurochemical markers have been identifiedthus far that distinguish between these two nuclei of the basal gangliain rodents. However, during prenatal and early postnatal development,neurogenetic gradients within different regions of the developingstriatum correspond to characteristic gradients in the caudate andputamen in animals where these nuclei are anatomically discrete (Bayer,Intl. J. Devel. Neurosci. 2:163-175, 1984). In rodents, new striatalneurons rostral to the decussation of the anterior commissure are addedin a lateral-to-medial gradient such that the latest born neurons arethose nearest the lateral ventricle. That same pattern is observed inthe development of the caudate nucleus, suggesting that the anteriorstriatum in rodents is more of a “caudate-like” region. Caudal to thecrossing of the anterior commissure, neurons are added in amedial-to-lateral gradient, similar to the developing putamen in animalswhere it is anatomically distinct. Thus, the posterior rodent striatummay be more “putamen-like”. The possibility was considered that thestriatal ridge, then, might represent a previously unrecognized borderbetween these two regions of rat striatum that allowed a dense buildupof cells, perhaps due to some neurochemical difference.

[0175] A third possibility for the source of the striatal ridge was alsoexamined. Explanted cells from the subependymal zones of the forebrainlateral ventricles of adult mammals have been found capable ofproliferating and differentiating into new neurons and glia,particularly when cultured in the presence of EGF-family neurotrophicfactors, including TGFα (Reynolds et al., J. Neurosci. 12:4565-4574,1992; Morshead et al., Neuron 13:1071-1082, 1994). Recently, EGF or TGFαinfused into the lateral ventricle stimulated proliferation of“EGF-responsive” stem and neural progenitor cells in the adult mousebrain (Craig et al., J. Neurosci. 16:2649-2658, 1996). A possibility forthe source of the striatal ridges in the present studies was that theTGFα infusions stimulated similar proliferative activity in the brainsof rats. Additionally, the possibility that the striatal ridges weremass migrations of proliferating neural progenitor cells derived fromthe subependymal regions was considered.

[0176] The experiments described below served to allow characterizationof this anomalous striatal ridge using a variety of histochemical andimmunohistochemical techniques. The origin of the ridge and the factorsinfluencing its appearance were also investigated by examining the timecourse of its formation in the striatum and by altering the combinationsof surgical and chemical treatments.

[0177] A. Experimental Protocols

[0178] Adult male Sprague-Dawley rats weighing 250-300 grams were usedthroughout the study. Twenty-four animals received standard midstriatalinfusions of rat TGFα (0.5 μg/day; Sigma Chemical Co.). Another 26 ratsreceived either artificial cerebrospinal fluid (aCSF) or no infusion. Asubset of animals in the standard TGFα infusion group and the controlgroup received stereotaxic 6-OHDA injections into the substantia nigra48 hours after the infusions were begun. Animals used in this portion ofthe study were classified into six groups according to theirinfusion-lesion combination, as follows: TGFα infusion, lesion (n=13);TGFα infusion, no lesion (n=11); aCSF infusion, lesion (n=12); aCSFinfusion, no lesion (n=9); no infusion, lesion (n=1); no infusion, nolesion (n=4).

[0179] Additional animals received TGFα infusions into other regions ofstriatum, the lateral ventricle, cerebral cortex, or the septum. Fourmore animals (two per group) received midstriatal TGFα infusions atone-half or one-tenth the standard dose. Also, two animals receivedmidstriatal infusions of epidermal growth factor (EGF) instead of TGFα.The EGF administered in these rats was at the standard 0.5 μg/day dose.All of the animals in these extra groups were lesioned. Rats in all ofthe experiments were typically perfused one to 16 days postlesion (3-18days of infusion). To determine whether the ridge would persist afterthe infusions ceased, the minipumps were removed from four animals withTGFα infusions at the end of two weeks, but these animals were notperfused until several days later. Brain sections were prepared andstained using various immunocytochemical and histochemical techniques.

[0180] B. TGFα Infusion

[0181] Rats were anesthetized with 8 mg xylazine and 100 mg ketamine/kg.Infusions of TGFα were provided up to 18 days by Alzet osmotic minipump(2002). The minipumps were filled to about 200 μl with either aCSF forcontrol animals or 20 μg TGFα in 400 μl of aCSF (50 μg/ml) forexperimental animals. Under sterile conditions, the infusion cannula waspositioned to stereotaxic coordinates (+1.2 A/P; +2.7 M/L; −6.0 D/V)based on Bregma (Paxinos and Watson, The Rat Brain in StereotaxicCoordinates, Academic Press, San Diego, 1986) and cemented to the top ofthe skull with dental cement. The infusate was delivered via cannula atapproximately 0.5 μl/hour. Some additional control animals receivedinfusions either into the lateral ventricle, the overlying cortex, orother areas.

[0182] C. Neurotoxic Lesion

[0183] Forty-eight hours after the minipump implant, rats wereanesthetized as above. A chilled 4.8 mg/ml solution of 6-OHDA HCl wasprepared immediately prior to injection. Using sterile technique, theneurotoxin was stereotaxically injected into the ipsilateral substantianigra (+3.7 A/P; +2.1 M/L; +2.0 D/V) using interaural zero as areference (Paxinos and Watson, 1986, supra). A 6-8 μl volume wasinjected at a rate of 1 μl/minute.

[0184] D. Tissue Preparation

[0185] Animals were perfused with 500 ml of 4% paraformaldehyde in 0.1 Mphosphate buffered saline (PBS; pH 7.4) one to 16 days postlesion andtheir brains placed into 20% sucrose. The next day, the brains werefrozen in isopentane at −20° C. Forty-micron coronal sections were thencut on a freezing microtome into 2% paraformaldehyde in 0.1 M PBS.Continuous sections were taken through the striatum and substantianigra-VTA. Representative sections were taken through the rest of thebrain.

[0186] E. Nissl Staining

[0187] Microtomed brain sections for Nissl staining were mounted ontogelatin-coated slides and allowed to dry overnight. They were thendehydrated and rehydrated through ethanol baths, and placed in Thioninsolution for approximately four minutes. The sections were dehydratedthrough the series of ethanol baths, cleared in successive Histoclearwashes, and coverslipped. Section were viewed under light microscopy andphotographed with Technical Pan Film (Kodak, Inc.) at ISO 100 (HC-110processing for six minutes).

[0188] F. Silver Staining

[0189] Degenerating fibers and cells of the ridge were labeled using amodification to the Nauta staining method, similar to Procedure I ofFink-Heimer (Giolli and Karamanlidis, In: Neuroanatomical ResearchTechniques, R. T. Robertson, Ed., pp. 211-240, Academic Press, New York,1978). Briefly, free-floating sections were placed into 0.05% potassiumpermanganate prior to treatment with fresh 1% hydroquinone-1% oxalicacid. They were treated with successive uranyl nitrate/silver nitratesolutions of increasing concentration. After another rinse, the sectionswere reacted in ammoniacal silver, then in ethanol/citricacid/paraformaldehyde reducer, and finally in sodium thiosulfate. Afterstaining, the sections were mounted on glass slides and allowed to dryon a slide dryer for 15 minutes. The sections were then dehydratedthrough successive ethanol washes of increasing concentration, defattedin three successive Histoclear washes, and coverslipped.

[0190] G. Immunohistochemistry

[0191] The quality and the extent of the nigral lesion were determinedby the loss of TH-IR in the ipsilateral ventral midbrain. Antibodiesagainst glial fibrillary acidic protein (GFAP), a marker for astrocytes,nestin, a marker for neural progenitor cells, and vimentin, a marker forradial glial cells, were employed in the neurochemical characterizationof the ridge. Immunohistochemistry was performed on free-floatingsections. Briefly, brain sections were washed in 0.1 M PBS or Trisbuffered saline (TBS; 3×10 minutes) then incubated for 1 hour inblocking solution consisting of 3% normal goat serum in 0.1 M PBS or TBSwith 250 μl Triton X-100. Next, they were incubated overnight at roomtemperature on a rotator with antibody solution diluted with blockingsolution: rabbit anti-TH antiserum (1:500; Eugene Tech Intl., Inc.),rabbit anti-GFAP (1:6400; Dako Corp.), mouse monoclonal anti-vimentin(1:50; Sigma Chemical Co.), or with mouse anti-nestin supernatant (1:20;University of Iowa Hybridoma Bank).

[0192] The sections then were washed and incubated for 1 hour withbiotinylated goat anti-rabbit antiserum (1:200; Vector Labs, Inc.) forTH or GFAP immunostaining, or biotinylated horse anti-mouse antiserum(Vector Labs, Inc.) for nestin or vimentin immunostaining, then washedand incubated in avidin-biotin complex (ABC Elite kit, Vector Labs,Inc.) for 1 hour. Localization of primary antibody binding was revealedusing the diaminobenzidine (DAB) peroxidase technique. The sections werewashed thoroughly and mounted on gelatin-subbed slides and allowed todry overnight. Finally, the sections were dehydrated, cleared, andcoverslipped as described above.

[0193] None of the animals used in the study displayed adverse effectsfrom the minipump implants or lesion surgeries. All continued to takefood and water through the course of the experiments. If the lesion,infusion, or both were not successful, the animals (n=6) were excludedfrom the initial experimental groups and examined separately. Asuccessful lesion was defined as one that caused complete ornear-complete elimination of ipsilateral nigral TH-IR. A successfulstriatal infusion was defined as one where the tip of infusion cannulawas successfully fixed into the body of the striatum.

[0194] As with the in situ hybridization studies, all animals receivingintrastriatal TGFα infusions of six days or more displayed a dramaticbuildup of cells along the ventricle ipsilateral to the infusion,visible with thionin staining. By comparison, the contralateral striatumshowed no such increase, and was indistinguishable from that inaCSF-infused animals. EGF infusions in lesioned animals induced thecellular expansion along the ventricle, but did not induce formation ofthe striatal ridge. Lower doses of TGFα induced both the cellularexpansion and ridge formation, but, qualitatively, the number of cellsin each was decreased.

1. Effects of 6-OHDA Lesions

[0195] None of the lesioned animals receiving aCSF infusions showed anycellular expansion along the ipsilateral ventricle or any evidence of astriatal ridge. Lesioned animals infused with TGFα did uniformly exhibitthe buildup of cells along the ventricle and typically displayed thestriatal ridge. Nigral lesions dramatically increased the incidence offormation of the ridge compared to unlesioned animals (Table 1).

2. Morphology and Persistence of the Ridge

[0196] Midstriatal infusion resulted in a characteristic S-shaped ridgearising from the dorsomedial caudate-putamen, sweeping out into thestriatum and looping back slightly toward the midline at its ventralend. The dorsal-most portion of the ridge was continuous with thebuild-up of cells in the subependymal region. Typically, thioninstaining was most dense in the dorsal portion of the ridge parallelingthe EGF receptor mRNA hybridization. The cellular ridge was generallyfound throughout most of the rostral-caudal extent of the striatum. Theridge was still prominent in the striatum three months after the TGFαinfusion pump was removed. Infusate Lesion Number Expansion aCSF no 9 0%0% aCSF yes 12 0% 0% TGFα no 11 100% 27% TGFα yes 13 100% 92%

3. GFAP Immunohistochemistry

[0197] Antiserum against glial fibrillary acidic protein (GFAP), amarker for astrocytes, failed to stain cells of the striatal ridge orthe cellular expansion along the ventricle. Normal GFAP-IR astrocyticstaining was found medial and lateral to the ridge, but was nearlyexcluded from the ridge itself.

4. Silver Staining

[0198] Labeling cells non-specifically with a modification of the Nautamethod provided additional information about the cells comprising theventricular cellular expansion and the striatal ridge. One of the moststriking features was the huge number of cells making up thesubependymal cellular buildup and ridge (FIG. 8). The cells were denselypacked and predominantly fusiform in shape (FIG. 8). In the ventralportion of the ridge, elongated cells appeared to stream around fiberbundles of the internal capsule suggesting that the cells were migratingthrough the striatum.

5. Time Course of Ridge Formation

[0199] The density of cells of the ridge allowed us to track itsformation using simple thionin staining (FIG. 9). All animals used forthe time-course experiment received 6-OHDA lesions and midstriatal TGFαinfusions. At time points prior to six days of infusion, there was onlya very minor build-up of cells in the subependymal region and noevidence of a striatal ridge. By six days of infusion, there was a clearexpansion of cells along the ventricle. At nine days infusion, theventral portion of the ridge had begun to appear slightly displaced fromthe ventricle. By 12 days infusion, the ventral portion of the ridge wassituated as much as 400 μm from the ventricle wall. At 16 days infusion,the ridge appeared midstriatum, its ventral portion up to two mm fromthe ventricle. Thus, the ridge originated in the ventricular region andwas increasingly displaced radially in the overlying striatum at greatertimes of infusion. The estimated difference in distance between thelateral extents of the ridges and the ventricle wall at 12 days and 16days of infusion was approximately 1.6 mm.

6. Nestin Immunohistochemistry

[0200] Monoclonal antibodies against nestin, a marker forneuroepithelial progenitor cells, intensely stained dense collections offibers throughout the ridge (FIG. 10). No nestin-IR fibers were seenlateral to the ridge, but occasional fibers were observed medial to theridge. The fibers were oriented primarily orthogonal to the ridge.

7. Alteration of Ridge Morphology

[0201] Lesioned animals infused midstriatally with TGFα uniformlyexhibited a characteristic S-shaped striatal ridge in coronal sections.This morphology was dramatically altered in rats with infusions intoother areas of the caudate-putamen (FIG. 11). Medial striatal infusionsgave rise to an L-shaped ridge near the cannula infusion site with thevertical part of the “L” along the ventricle and the horizontal partextending orthogonally from the ventricle into the striatum. Infusioninto the extreme lateral striatum stimulated the formation of a linearridge parallel to the wall of the lateral ventricle.

8. Vimentin Immunohistochemistry

[0202] Antiserum recognizing vimentin, a marker for radial glial cells,failed to stain any cells in the striatum, the subependymal zone, or thestriatal ridge at two weeks of infusion. However, this result is beinginvestigated further, as the controls were performed in embryonicanimals, under conditions that may not ensure elimination of falsenegatives.

9. Control of Ridge Position

[0203] Compared to the cells of ridges in rats used for in situhybridization experiments, ridge cells in the present series ofexperiments were maximally displaced much farther from the ventricle(FIG. 12). The difference between these two groups of animalsexperimentally was the timetable of infusions and lesions.

[0204] Animals prepared for in situ hybridization experiments receivedlesions and then underwent a series of behavioral tests starting aroundthe second week postlesion to confirm success of the unilateral lesions.Typically, those animals did not receive infusions until five weeksafter the lesion. Thus, the dopamine degenerative process and thestriatal infusion of TGFα were temporally separate events.

[0205] In the present series of experiments, infusions were begun first;lesions were not performed until 48 hours after the infusion pumps wereimplanted. In these animals, the degeneration of the nigral dopamineneurons, the resulting loss of striatal dopaminergic innervation, andthe striatal administration of growth factor were temporally concurrentevents.

10. Infusions Into Brain Regions Other Than the Striatum

[0206] All rats receiving infusions into brain areas other than thestriatum received two-week TGFα infusions and nigral 6-OHDA lesions.Intracerebroventricular (ICV) infusion of growth factor ipsilateral tothe lesion stimulated the buildup of cells in the adjacent ventricularwall, but did not induce formation of the striatal ridge in any of theanimals.

[0207] Septal and some striatal infusions stimulated the formation ofseptal ridges associated with the medial walls of the lateralventricles. Septal ridges, like the striatal ridges, were readilydetected with EGF receptor mRNA in situ hybridization or with thioninstaining, but tended to be qualitatively less robust in terms of thedensity and number of cells.

[0208] Dorsal cortical infusions, that is, infusions so shallow thatthey did not penetrate the corpus callosum, had no discernable effect oncell density along the lateral ventricle. Neither did these dorsalinfusions induce formation of a ridge. Cortical infusions in which thecorpus callosum was slightly penetrated stimulated expansion of cellsalong the ipsilateral ventricle, but did not induce formation of astriatal ridge. These animals did exhibit densities of cells in thecorpus callosum that might be considered callosal ridges.

H. TGFα Stimulates Cellular Proliferation

[0209] The present experiments, together with those described above,demonstrate that TGFα administration was necessary for the formation ofthe cellular build-up and the striatal ridge. Not a single animal thatreceived a striatal aCSF infusion—whether lesioned or not—displayed anyobvious periventricular cellular expansion when compared to subependymalregions contralateral to the infusions or to normal animals. Clearly,cells of the forebrain are responding to striatal infusion of TGFα byproliferating along the lateral ventricle.

[0210] Recent studies with 6-day ICV infusions of EGF or TGFα in micedemonstrated a large increase in the number of cells around theventricle immunolabeled with 5-bromo-2′-deoxyuridine (BrdU) or[³H]thymidine, markers for cellular proliferation (Craig et al., J.Neurosci. 16:2649-2658, 1996). More than 95% of these cells were alsopositively immunoreactive for EGF receptor. Cresyl violet Nissl stainingalso showed an increase in the numbers of cells around the ventricles inthese animals in response to growth factor administration.

[0211] The possibility that the expanded cell populations along thelateral ventricles were glial cells stimulated by the combinedneurotoxic lesion and mechanical injury of the forebrain, and theinfused TGFα was considered first. Astrocytes are known to respond tobrain injury by proliferating and altering their morphology andfunctional properties (for review see Norenberg, J. Neuropath. Exper.Neurol. 53:213-220, 1994). Additionally, striatal astrocytes possess EGFreceptors (Gómez-Pinilla et al., 1988, supra; Nieto-Sampedro et al.,1988, supra) and are stimulated by TGFα to proliferate (Alexi and Hefti,1993, supra). In the present study, antiserum against glial fibrillaryacidic peptide (GFAP), a marker for astrocytes, failed to demonstrate anincrease in astrocytes in the ventricular region or in the ridge at twoweeks of infusion. In fact, GFAP-IR was largely excluded from theseareas. Normal astrocytic staining was seen medial and lateral to theridge, for instance, but few GFAP-IR fibers were observed within theridge itself. These findings paralleled those in experiments withsix-day ICV infusions of EGF or TGFα: GFAP and an additional marker forastrocytes, S100β, showed no significant increase around the lateralventricle (Craig et al., 1996, supra). Vimentin can also be expressed byreactive astrocytes (Federoff et al., J. Neurosci. Res. 12:14-27, 1984),but vimentin-IR was not observed in any of the sections examined.Markers for microglia (MAC-1) and oligodendrocytes (MAG, CNP, O4 andRip) also did not change significantly (Craig et al., 1996, supra).Thus, the immunohistochemical evidence demonstrated that theTGFα-induced expansion of cells along the ventricle and in the striatalridge were not the result of gliosis.

1. Cellular Morphology and Orientation

[0212] Silver and thionin staining clearly reveal the huge numbers ofcells within the cellular aggregation along the ventricle. The cellswere most dense and most numerous in the dorsal portions of thesubependymal zone and ridge. The cells were predominantly fusiform,similar to migrating neural progenitor cells in the developing brain,with their long axes oriented orthogonal to the ventricle wall (or tothe dorsolateral extension of the subependymal zone bordering the dorsalstriatum). Silver stained cells in the ventral segment of the ridgeappeared to stream around fiber bundles of the internal capsule,suggesting that they were migrating through the striatum.

[0213] To determine whether the cells were indeed migrating, a timecourse experiment was performed to examined the development of the ridgeand its location as a function of time after the start of the growthfactor infusion.

2. Migration of Cells of the Striatal Ridge

[0214] The progressive expansion of cells along the lateral ventricleand the subsequent radial movement of these cells as a dense ridgeproved that the cells were indeed migrating en masse through thestriatum. As such, the ridge could not have been an anatomicaldelineation between the rodent putative “caudate-like” and“putamen-like” regions of striatum.

3. Stimulation of Neural Precursor Cells

[0215] Although none of the immunomarkers for mature astrocytes,neurons, microglia, or oligodendrocytes labeled cells of theperiventricular expansion or the striatal ridge, monoclonal antibodiesrecognizing nestin, an intermediate filament expressed byneuroepithelial precursor cells, intensely stained cell processes in theridge and along the ventricle. Reactive astrocytes can also expressnestin-IR, but the negative GFAP-IR in cells of the ridge eliminated thepossibility that astrocytes formed a significant portion of the ridgecells. Nestin-IR has been used in recent years to identify and labelneural precursor cells in vitro and in vivo (Lendahl et al., Cell60:585-595, 1990; Craig et al., 1996, supra). The strong nestin-IR inthe striatal ridge and the lack of immunostaining for glial markerssupport the conclusion that the cells of the ridge are predominantlyneural progenitor cells.

[0216] Thus far, two distinct cell populations have been identified inadult mammalian brain that can give rise to new neurons and glia(Morshead et al., 1994, supra). One, the relatively quiescent cellpopulation, are believed to be true multipotent neural stem cells. Theother, the constitutively proliferating population, are believed to beneural progenitor cells, descendent from the stem cells. The stem cellpopulation is thought to remain in the ependymal or subependymal zoneand replenish the progenitor cell population as they die or migrateaway. The term “neural precursor” is used here to describe anyundifferentiated proliferative cell capable of giving rise to neuronsand glia in the adult mammalian brain, whether these cells are neuralstem cells or neural progenitors.

[0217] Previous studies have shown that many neural progenitor cells diein the subependymal zone before they can migrate from the region(Morshead and Van der Kooy, J. Neurosci. 12;249-256, 1992). However, itwas recently discovered that many others indeed survive and migratealong a highly-restricted path to the olfactory bulbs where theydifferentiate into olfactory interneurons (Luskin, Neuron 11:173-189,1993; Lois and Alvarez-Buylla, Science 264:1145-1148, 1994). Theymigrate tangentially along the wall of the lateral ventricle in aprocess called “chain migration” wherein chains of migrating cells areensheathed by specialized GFAP-IR astrocytes (Rousselot et al., 1994;Lois et al., Science 271:978-981, 1996).

[0218] The subependymal zone along the forebrain lateral ventricles,then, is much more than a dormant remnant of the embryonicneuroepithelium. In normal unmanipulated brains, it continues to giverise to new neuroblasts that migrate rostrally and differentiate intoneurons. Under the influence of EGF-family neurotrophic factors,including TGFα, subependymal neural precursors can be stimulated invitro to give rise to large numbers of new neurons, astrocytes andoligodendrocytes (Reynolds and Weiss, Science 255:1707-1710, 1992). Fromthese explant studies, it became clear that the highest concentrationsof neural precursor cells were found in the dorsal portion of thesubependymal zone, along the dorsal border of the caudate-putamen.

[0219] There is even some recent evidence that neural precursors may bestimulated to increase their numbers and produce new neurons and glia invivo (Craig et al., 1996, supra). Cells double-labeled with BrdU andmarkers for mature neurons and glia were found diffusely distributedthroughout the striatum, septum, and cortex after six days of ICVinfusion of EGF and up to seven weeks of post-infusion survival.However, no mass migration of subependymal cells into the adjacentstriatum was observed in that study (in marked contrast to the resultsobtained by the present method) and the numbers of cells were quitemodest compared to the densely-packed mass of cells observed in thestriatal ridge described herein. Moreover, none of the animals in Craiget al. received an infusion sufficient to stimulate the mass migrationpresently observed and none of the animals in that study received nigral6-OHDA lesions, which are shown for the first time herein todramatically increase the incidence of migration.

4. Evidence of Neuronal Phenotype

[0220] A question that remained was whether the cells within the massiveexpansion along the ventricle and the migrating striatal ridge trulywere neural progenitor cells. Table 2 summarizes the data supporting theconclusion that these cells are indeed neural progenitors. Theneurochemical evidence showed that they do not express markers formature neurons, astrocytes, oligodendrocytes, or microglia. They did,however, intensely express a marker for immature neural progenitorcells. They expressed markers for cellular proliferation. They arosefrom the wall of the lateral ventricle where neural precursors arelocated in the adult rodent brain, expanded laterally and migratedradially away from the ventricle. Furthermore, their cellular morphologywas fusiform and their processes were oriented orthogonal to theventricle and the ridge, similar to migrating neural progenitors in theembryonic brain and consistent with their migration from thesubependymal region. The data summarized in the following Table (Table2) indicates that cells of the periventricular expansion and thestriatal ridge are neural progenitors arising from subependymal neuralstem cells. Evidence of Progenitor Phenotype for Cells of the Expansionand Ridge Neurochemical Express abundant EGF receptor mRNA* andimmunoreactivity Immunonegative for GFAP* or S-100β markers forastrocytes Immunonegative for NeuN, a marker for mature neuronsImmunonegative for MAG, CNP, 04, or Rip, markers for oligodendroctyesImmunonegative for vimentin*, a marker for radial glia Immunonegativefor MAC-1, a marker for microglia Immunopositive for nestin*, a markerfor neuroepithelial precursors Morphological Elongated somata in ridgeoriented orthogonal to subependymal zone Nestin-IR processes in ridgeoriented normal to subependymal zone Anatomical Arise from thesubependymal zone Highest density of cells is in the dorsal subependymalregion Physiological Respond to TGFα administration by increasing theirnumbers

5. Mechanisms of Migration

[0221] The mass migration of these cells into the striatum could becontrolled by striatal dopamine denervation and by the location of theinfusion cannula, but the mechanism of migration was unclear. The factthat the shape of the ridge could be modified simply by changing thesite of infusion initially suggested a chemoattractant effect. TGFα isknown to be a potent chemoattractive agent for diverse cell types (Ju etal., J. Invest. Dermatol. 100:628-632, 1993; Panagakos, Biochem. Mol.Biol. Int. 33:643-650, 1994). Its abundant expression in the perinatalcaudate-putamen may indicate that it performs a similar role in thedevelopment of the striatum.

[0222] Neural precursor cells in the normal brain are located in a thinregion in the wall of the lateral ventricle. Infusions into themid-striatum were closer to cells of the dorsal end of this region.Presumably, cells migrating into the striatum would move toward putativehigher concentrations of growth factor at the tip of the infusioncannula where the TGFα was released. The characteristic S-shape ofridges in animals with mid-striatal infusions might have resulted fromreceptor saturation of cells in the dorsal segment of the subependymalzone nearest the tip of the infusion cannula. Cells with saturated EGFreceptors might have halted their migration once they moved close to theinfusion site. Cells near the ventral end of the subependymal zone wouldsee a lower putative growth factor concentration and would have totravel farther toward the infusion site before the concentration of TGFαincreased enough to saturate their receptors. This differentialmigration with receptor saturation could explain the characteristicS-shape of these ridges.

[0223] Infusions into the medial striatum resulted in L-shaped ridges,again in keeping with a neurochemical gradient/receptor saturationeffect. In this instance, dorsal subependymal cells may have had theirreceptors saturated and their migration halted before they could evenemerge from the subependymal zone. Only cells in the most ventralportion of the subependymal region could migrate away from theventricle.

[0224] Extreme lateral infusions essentially would have presented asimilar putative concentration gradient to cells along most of thelength of the proliferative region. The subepenpdymal cells all migrateda similar distance from the ventricle, resulting in a roughly linearridge, consistent with the idea of a chemoattractant, neurochemicalgradient effect.

[0225] However, immunohistochemical evidence from the characterizationstudies cast doubt on the idea that a simple chemoattractant effectcould entirely explain the mass radial migration. Nestin-IR processes ofthe migrating cells were not aligned with the tip of the infusioncannula, the region of the putative highest concentration of growthfactor. Instead, they were oriented normal to the ventricle and theridge. This orientation suggested that the cells migrated orthogonallyinto the caudate-putamen—as migrating neural progenitors do from theembryonic striatal neuroepithelium—not obliquely toward the tip of theinfusion cannula.

[0226] Two additional findings discounted the role of simplechemoattraction in the migration of the ridge. First, the cells did notbegin to migrate as they were produced; they increased their numbersalong the ventricle for a period of more than a week, then migrated enmasse as a dense sheet of cells. Further, lesion of the ipsilateralsubstantia nigra greatly increased the incidence of migration. Both ofthese observations suggested a more complex set of factors influencingthe migration of the cells. These data did not entirely rule out a rolefor chemoattraction in the migration of the ridge, but they did indicatethat a simple chemoattractive effect could not by itself account for it.

[0227] Another mechanism that might have contributed to the radialmigration of neural progenitors in the adult striatum was thereconstruction of the radial glial scaffold due to the neurotoxiclesion, the mechanical injury done by the surgical implantation of theinfusion cannula, or both. Radial glia guide the migration of neuralprogenitor cells in many regions of developing brain. They are anchoredalong the ventricle and extend their processes radially into theoverlying parenchyma. They normally are transformed into GFAP-IRastrocytes in the early postnatal period once neuroblast migration iscomplete, and cease expression of vimentin and nestin.

[0228] Freezing injury of the cortical plate in neonatal rats inhibitedradial glial transformation and caused the persistence of glialexpression of vimentin and nestin in the injured regions of the adultbrain (Rosen et al., Dev. Brain Res. 82:127-135, 1994). Kainate lesionof the adult rat hippocampus induced radial glial morphology andexpression of nestin-IR and vimentin-IR in astrocytes of the hippocampalsubependymal zone, suggesting that brain injury could stimulate areversion of astrocytic phenotype to one found in the embryonic brain(Clarke et al., Neuroreport 5:1885-1888, 1994).

[0229] The present immunohistochemical experiments do not providesupport for this phenomenon. Nestin-IR fibers were found in abundance inradially-oriented fibers along the ventricle at infusion day nine, butat later time points, they no longer remained along the ventricle. Asthe cells of the ridge migrated away from the subependymal region, sodid the nestin-IR fibers. Furthermore, immunostaining for vimentin, aspecific marker for radial glia, did not reveal any labeled fiberseither in the ridge or in the subependymal region. Thus, it is unlikelythat any astrocytes were reverted to their embryonic radial glialphenotypes or that astrocytic reversion played a role in the radialmigration of the neural progenitor cells.

[0230] A newly-described mode of migration employed specifically byneural progenitors in the adult mammalian brain elucidates thetangential movement of these cells from the forebrain subependymal zonesto the olfactory bulbs (Lois et al., 1996, supra). Rostrally migratingneuroblasts are densely packed and sheathed by GFAP-IR astrocytesbordering their highly-restricted migratory pathway. The migrating cellsessentially form a solid stream of moving cells within a tube ofspecialized glial guide cells. The neural precursors in our experimentsmigrated as a sheet through the striatal neuropil—not along a restrictedpath—and were not associated with GFAP-IR cells. In fact, theproliferative subependymal zone and the cellular ridge largely excludedGFAP-IR. Thus, the mechanism of chain migration could not account forthe radial migration seen in the present studies.

[0231] Another mechanism possibly underlying the mass neural progenitormigration was that cells of the striatum may have altered theirexpression of growth factors, cell adhesion molecules, or othersubstances in response to injury. In this scenario, the striatum mayhave been stimulated to provide its own chemoattractants or moleculesthat facilitate radial migration. Alternatively, it may also have beeninduced to downregulate expression of substances that inhibit migration.

[0232] Recent studies examining cell adhesion molecules in the striatumand subependymal region provide particularly intriguing insight. Highlypolysialylated neural cell adhesion molecule (PSA-N-CAM)immunoreactivity is intensely expressed in the developing rodentstriatum, but decreases as the animal matures (Aaron and Chesselet,Neurosci. 28:701-710, 1989; Szele et al., Neurosci. 60:133-144, 1994).PSA-N-CAM expression, however, persists along the adult forebrainsubependymal region (Rousselot et al., 1994; Szele et al., 1994, supra).Partial decortication-induced striatal deafferentation dramaticallyincreased expression of PSA-N-CAM and another adhesion molecule, L1, inthe subependymal zone (Poltorak et al., J. Neurosci. 13:2217-2229, 1993;Szele and Chesselet, J. Comp. Neurol. 368:439-454, 1996). In the humanbrain, PSA-N-CAM expression is low in normal striatum, but is increasedin the striata of Huntington's disease patients, particularly in thesubependymal zone (Nihei and Kowall, Ann. Neurol. 31:59-63, 1992).

[0233] Of special interest were the changes occurring in fibronectinmRNA expression in the striatum after partial unilateral frontaldecortication (Popa-Wagner et al., Neuroreport 3:853-856, 1992).Fibronectin is one of a number of molecules that have been shown tosupport neural migration in vitro (Fishman and Hatten, J. Neurosci.13:3485-3495, 1993). Fibronectin mRNA hybridization was increased to amaximum level at 72 hours in the portion of the striatum immediatelyunder the wound cavity. This early increase was interpreted as acomponent of the short-term wound healing process. Fibronectinexpression in the greater ipsilateral striatum followed a longer-termincrease, peaking at about ten days post lesion. This secondary increasewas interpreted as the striatal response to deafferentation. Increasesin expression of two other mRNAs that code for N-CAM and alpha tubulinfollowed only the early wound healing-related spatial and temporalpatterns.

[0234] The ten-day peak of striatal fibronectin mRNA expression afterdeafferentation corresponds well to the delay of ridge migrationfollowing striatal dopamine denervation in the present studies. Thedelay of peak fibronectin mRNA expression may help explain why theprogenitor cells of the invention did not begin to migrate radiallyuntil around the ninth day of infusion, and why, when they finally didmigrate, they migrated en masse. In animals where the infusions andlesions were separated by several weeks, the maximum lateral migrationdistances at two weeks of infusion were dramatically reduced. Thisobservation, too, is consistent with the transient peak in striatalfibronectin expression. In the few animals with ridges that did not alsoreceive nigral lesions, mechanical injury of the overlying cortex mayhave stimulated enough fibronectin expression in the striatum tofacilitate the migration. Fibronectin, then, may be upregulated inresponse to dopamine denervation of the ipsilateral striatum and, inturn, may temporarily facilitate radial migration of neural progenitorsfrom the subependymal zone.

[0235] Another possible influence on radial migration of neuralprogenitors in the adult striatum may stem from a secondary effect bycell adhesion molecules. Infusion of N-CAM into the brains of adult ratsreceiving stab wounds to various areas of the brain, including striatum,inhibited astrocytic proliferation (Krushel et al., Proc. Natl. Acad.Sci. USA 92:4323-4327, 1995). Astrocytes release factors inhibitingneurite outgrowth and may thus inhibit neural regenerative responses.Thus, denervation of the striatum, and the associated increase insubependymal PSA-N-CAM may release inhibition of neural regeneration.

[0236] The enhancement of the migration effect in dopamine-denervatedstriatum may also have been related directly to the loss of dopaminergicinnervation. During embryonic development of the striatum, immatureneurons originate in the ventricular region and migrate radially (Bayer,1984, supra; Bayer and Altman, Prog. Neurobiol. 29:57-106, 1987). Thedevelopmental migration of striatal neurons takes place prior todopamine innervation by afferents from the midbrain. Stimulation of D₂dopamine receptors on hypothalamic neurons dramatically attenuated TGFαmRNA expression and pituitary growth (Borgundvaag et al., Endocrinology130:3453-3458, 1992). Although dopamine receptor-mediated inhibition ofTGFα expression has not been studied in the subependymal zone, it isconsistent with the depressed incidence of migration in non-lesionedanimals. Thus, dopamine innervation during development may inhibitmigration of striatal cells as the forebrain dopaminergic innervationbecomes established.

[0237] Striatal dopamine may also contribute to the downregulation ofstriatal TGFα early in postnatal development as dopaminergic afferentsbecome established. Dopamine denervation of the adult striatum may mimicfor striatal cells some of the local chemical environmental cuesnormally only seen in the developing striatum—for instance, a reductionof available ligand for dopamine receptors expressed on striatalneurons. Striatal dopamine innervation has also been linked in areciprocal manner to expression of extracellular matrix (ECM) moleculesby astrocytes (Gates et al., J. Chem. Neuroanat. 6:179-189, 1993).Interestingly, TGFα is selectively elevated in the striata of PDpatients (Mogi et al., Neurosci. Lett. 180:147-150, 1994) similar to theelevated expression in the embryonic striatum. If striatal TGFα isregulated by dopamine innervation, this increase may relate to thereduction of striatal dopamine and the consequent release of inhibitionof TGFα expression.

[0238] Whatever the underlying mechanism, the time course experimentproved that subependymal cells could be stimulated to increase theirnumbers and migrate radially en masse into the adjacent striatum in theadult rat brain. Experiments in which the location or the dose of TGFαinfusion was varied showed that movement of the striatal ridge and thegross numbers of cells involved could be controlled. Thecharacterization experiments provided abundant evidence that thesubependymal cellular expansion and the dense striatal ridge werecomposed of neural progenitor cells. The importance of these discoveriesis discussed below, together with their potential application for thetreatment of human neurodegenerative disease and traumatic brain injury.

[0239] The invention has now been explained with reference to specificexamples and embodiments. Other embodiments will be suggested to thoseof ordinary skill in the appropriate art upon review of the presentspecification.

[0240] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for treating a patient who has aneurological deficit, the method comprising (a) contacting a neuralprogenitor cell of the patient's central nervous system (CNS) with apolypeptide that binds the epidermal growth factor (EGF) receptor, thedosage of the polypeptide being sufficient to stimulate theproliferation of the neural progenitor cell, and (b) directing progenyof the proliferating progenitor cell to migrate en masse to a region ofthe CNS in which the cells will reside and function in a mannersufficient to reduce the neurological deficit.
 2. The method of claim 1,further comprising contacting the cells with a compound that stimulatesthe progeny of the proliferating neural progenitor cells todifferentiate.
 3. The method of claim 1, wherein the neurologicaldeficit is caused by a neurodegenerative disease, a traumatic injury, aneurotoxic injury, ischemia, a developmental disorder, a disorderaffecting vision, an injury or disease of the spinal cord, ademyelinating disease, an autoimmune disease, an infection, or aninflammatory disease.
 4. The method of claim 3, wherein theneurodegenerative disease is Alzheimer's Disease, Huntington's Disease,or Parkinson's Disease.
 5. The method of claim 3, wherein the ischemiais associated with a stroke.
 6. The method of claim 1, wherein thepolypeptide that binds the EGF receptor is amphiregulin (AR),betacellulin (BTC), epidermal growth factor (EGF), epiregulin (ER),heparin-binding EGF-like growth factor (HB-EGF), schwannoma-derivedgrowth factor (SDGF), myxomavirus growth factor Shope fibroma virusgrowth factor, teratocarcinoma-derived growth factor-1 (TDGF-1),transforming growth factor alpha (TGFα), or vaccinia growth factor(VGF).
 7. The method of claim 1, wherein the polypeptide that binds theEGF receptor is TGFα.
 8. The method of claim 1, wherein the neuralprogenitor cell is contacted in vivo with a polypeptide that binds theEGF receptor.
 9. The method of claim 1, wherein the neural progenitorcell is contacted in culture with a polypeptide that binds the EGFreceptor.
 10. The method of claim 1, wherein migration is directed bycontacting a cell along, or at the end of, a desired path of migrationwith a compound that increases the expression of a cell adhesionmolecule or extracellular matrix molecule.
 11. The method of claim 10,wherein the compound is TGFα.
 12. The method of claim 10, wherein thecell adhesion molecule is fibronectin.
 13. The method of claim 10,wherein the cell adhesion molecule is laminin.
 14. The method of claim10, wherein the compound is applied along the path between the neuralprecursor cells and the location to which their progeny are directed tomigrate.
 15. The method of claim 1, wherein migration is directed bycontacting the cells along a desired migratory path with a compound thatinhibits a naturally occurring signal along the path, the naturallyoccurring signal being a signal that inhibits migration.
 16. The methodof claim 1, wherein migration is directed by mechanically disruptingtissue in the CNS.
 17. The method of claim 1, wherein migration isdirected by neurochemically blocking the activity of cells in the CNS.18. The method of claim 2, wherein the compound that stimulatesdifferentiation is retinoic acid or brain-derived neurotrophic factor.19. A method for treating a patient who has a neurological deficit, themethod comprising (a) contacting a neural progenitor cell of thepatient's central nervous system (CNS) with a polypeptide that binds theepidermal growth factor (EGF) receptor, the dosage of the polypeptidebeing sufficient to stimulate the proliferation of the neural progenitorcell; (b) directing the progeny of the proliferating progenitor cells tomigrate en masse to a second region of the CNS; and (c) contacting thecells that have migrated with a compound that stimulatesdifferentiation.
 20. The method of claim 19, wherein the compound thatstimulates the proliferation of neural stem cells and the compound thatstimulates differentiation are administered sequentially.
 21. Apharmaceutical composition comprising a polypeptide that binds theepidermal growth factor (EGF) receptor and a compound that stimulatesthe differentiation of neural progenitor cells.
 22. The pharmaceuticalcomposition of claim 21, wherein the polypeptide that binds the EGFreceptor is TGFα.
 23. The pharmaceutical composition of claim 21,wherein the polypeptide that binds the EGF receptor is TGFα and thecompound that stimulates the differentiation of neural progenitor cellsis brain-derived neurotrophic factor.
 24. The method of claim 1, whereinthe injury to the central nervous system is an injury to the spinalcord.
 25. The method of claim 1, wherein the injury to the centralnervous system is an injury to the retina.
 26. A method for treating asubject having a neurological deficit, the method comprising contactinga neural precursor cell in vivo with a therapeutically effective amountof a polypeptide that binds the epidermal growth factor (EGF) receptor,wherein the polypeptide is parentally administered to the subject, andwherein the administration induces the proliferation, migration, ordifferentiation of a neural precursor cell in a manner sufficient totreat the neurological deficit.
 27. The method of claim 26, wherein theneurological deficit is caused by a neurodegenerative disease, atraumatic injury, a neurotoxic injury, ischemia, a developmentaldisorder, a disorder affecting vision, an injury or disease of thespinal cord, a demyelinating disease, an autoimmune disease, aninfection, or an inflammatory disease.
 28. The method of claim 27,wherein the neurodegenerative disease is Alzheimer's Disease,Huntington's Disease, or Parkinson's Disease.
 29. The method of claim27, wherein the ischemia is associated with a stroke.
 30. The method ofclaim 26, wherein the polypeptide that binds the EGF receptor isamphiregulin (AR), betacellulin (BTC), epidermal growth factor (EGF),epiregulin (ER), heparin-binding EGF-like growth factor (HB-EGF),schwannoma-derived growth factor (SDGF), myxomavirus growth factor Shopefibroma virus growth factor, teratocarcinoma-derived growth factor-1(TDGF-1), transforming growth factor alpha (TGFα), or vaccinia growthfactor (VGF).
 31. The method of claim 26, wherein the polypeptide thatbinds the EGF receptor is TGFα.
 32. The method of claim 26, whereinmigration is directed by contacting a cell along, or at the end of, adesired path of migration with a compound that increases the expressionof a cell adhesion molecule or extracellular matrix molecule.
 33. Themethod of claim 32, wherein the compound is TGFβ.
 34. The method ofclaim 32, wherein the cell adhesion molecule is fibronectin.
 35. Themethod of claim 32, wherein the cell adhesion molecule is laminin. 36.The method of claim 32, wherein the compound is applied along the pathbetween the neural precursor cells and the location to which theirprogeny are directed to migrate.
 37. The method of claim 26, whereinmigration is directed by contacting the cells along a desired migratorypath with a compound that inhibits a naturally occurring signal alongthe path, the naturally occurring signal being a signal that inhibitsmigration.
 38. The method of claim 26, wherein migration is directed bymechanically disrupting tissue in the CNS.
 39. The method of claim 26,wherein migration is directed by neurochemically blocking the activityof cells in the CNS.
 40. The method of claim 26, wherein differentiationis stimulated by retinoic acid or brain-derived neurotrophic factor. 41.A method for treating a subject having a neurological deficit, themethod comprising (a) contacting a neural precursor cell in vivo with atherapeutically effective amount of a polypeptide that binds theepidermal growth factor (EGF) receptor, wherein the polypeptide isparenterally administered in a dosage sufficient to stimulate theproliferation of the neural progenitor cell; (b) directing the progenyof the proliferating progenitor cells to migrate en masse to a secondregion of the CNS; and (c) contacting the cells that have migrated witha compound that stimulates differentiation.
 42. The method of claim 41,wherein the polypeptide that stimulates the proliferation of neural stemcells and the compound that stimulates differentiation are administeredsequentially.
 43. A pharmaceutical composition comprising a polypeptidethat binds the epidermal growth factor (EGF) receptor and a compoundthat stimulates the differentiation of neural precursor cells.
 44. Thepharmaceutical composition of claim 43, wherein the polypeptide thatbinds the EGF receptor is TGFα.
 45. The pharmaceutical composition ofclaim 43, wherein the polypeptide that binds the EGF receptor is TGFαand the compound that stimulates the differentiation of neural precursorcells is brain-derived neurotrophic factor.
 46. The method of claim 26,wherein the injury to the central nervous system is an injury to thespinal cord.
 47. The method of claim 26, wherein the injury to thecentral nervous system is an injury to the retina.